Polyamine compounds and compositions for use in conjunction with cancer therapy

ABSTRACT

The invention provides novel polyamine compounds and pharmaceutical compositions for administration in conjunction with cancer chemotherapy or radiation therapy. The compounds are administered locally to provide protection against the adverse side-effects of chemotherapy or radiation therapy, such as alopecia, mucositis and dermatitis. Pharmaceutical preparations comprising one or more chemoprotective polyamines formulated for topical or local delivery to epithelial or mucosal cells are disclosed. Methods of administering the pharmaceutical preparations are also disclosed.

[0001] This application is a continuation of U.S. application Ser. No.10/360,195, filed Feb. 7, 2003 which claims benefit of U.S. ProvisionalApplication No. 60/355,356, filed Feb. 7, 2002, the entirety of each ofthese applications is incorporated by reference herein.

[0002] Pursuant to 35 U.S.C. §202 (c), it is acknowledged that theUnited States Government has certain rights in the invention describedherein, which was made in part with funds from the National Institutesof Health, Grant No. CA22484.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of cancer therapy. Inparticular, the invention provides novel polyamine compounds andpharmaceutical compositions for reducing or preventing toxic sideeffects of radiotherapy and cancer chemotherapeutic agents.

BACKGROUND OF THE INVENTION

[0004] Various patents and other publications are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications is incorporated by reference herein, in its entirety.

[0005] It is well known that the use of chemotherapy and radiotherapy totreat cancer patients is associated with severe side effects due to thetoxicity of such treatments to epithelial cell populations, includingstem cells within the hair follicle, skin epidermis and gastrointestinalmucosa.

[0006] Currently, there are no treatments to prevent cancer therapy sideeffects. Effective treatments would likely include molecules that i)inhibit or slow growth of the at-risk cells, ii) modify the cellular DNAof the at-risk cells to make it less easily damaged, and iii) providesome means with which to scavenge electrophilic drug metabolites oroxygen radicals formed during irradiation.

[0007] Polyamines have been proposed as growth regulators. DENSPM, asynthetic analog of spermine, has been shown to decrease cell growth(Kramer et al., Cancer Res. 57:5521-5527, 1997), and has been studied inan early stage clinical trial as an antineoplastic drug (Creaven, P. etal., Invest. New Drugs 15:227-234, 1997; Streiff, R and Bender, J.,Invest. New Drugs 19:29-39, 2001). The clinical trials, however, wereaborted because of the serious side effects in multiple organ sites thatwere associated with the systemic use of this polyamine analog. Theseresults teach that molecules used to decrease division of healthy stemcells that are at risk from cancer therapy would need to create atransient cell cycle block and would need to be applied topically toachieve local delivery to epithelial cells, with little or no systemicdelivery, or if any, low enough to preclude protection of systemiccancer cells or induction of systemic side effects.

[0008] Naturally occurring polyamines, such as spermine, have been shownto bind to nucleic acids and to induce structural changes in helical DNA(Basu, H. and Marton, L., Biochem. J. 244:243-246, 1987; Feuerstein, B.et al., Nuc. Acids Res. 17:6883-6892, 1989). This binding has beensuggested to occur through interaction of the positively charged aminegroups in the polyamine backbone and negatively charged sites on the DNAbackbone. Because of the nature by which electrophilic chemotherapydrugs or oxygen radicals generated by radiotherapy attack helical B-DNAwithin cells, the ability of polyamines to bind DNA and disrupt normalB-DNA structure could be helpful in protecting DNA within cells to whicha polyamine was delivered.

[0009] An additional strategy for protecting cells againstelectrophiles/radicals has been to augment levels of the naturallyoccurring cellular nucleophile, glutathione (GSH). Both animal and cellculture studies have shown that there is a direct relationship betweenthe intracellular concentration of GSH and the amount of exogenouslyadministered alkylating molecule that is needed to achieve cell kill(Ho, D. and Fahl, W., J. Biol. Chem. 259:11231-11235, 1984;Ellouk-Achard, S. et al., Arch. Toxicol. Suppl. 17:209-214, 1995).Efforts to exogenously administer GSH to cells as a protectant havefailed because mammalian cells are generally unable to take up thisnucleophile. There have been efforts to modify the GSH molecule toenable cellular uptake, but these have not found clinical use.

[0010] Amifostine (WR-2721), a small molecule amine containing athiophosphate group that is presumably converted to a thiol in cells,has been used systemically as a radio- and chemoprotectant with mixedresults. Though it may provide free —SH groups within cells, it is notknown to contain activity as either a growth regulator or as a modifierof DNA structure.

[0011] Edwards et al. (U.S. Pat. No. 5,217,964 and U.S. Pat. No.5,434,145) described the synthesis of short, spermidine- orspermine-like polyamine molecules that were modified to contain analkyl-thiophosphate or alkyl-thiol group. In U.S. Pat. No. 5,217,964,the attached thiophosphate group (i.e., —SPO₃H₂) would require enzymaticactivation by cellular phosphatases to form the nucleophilic —SH group.The alkyl-thiophosphate group(s) is bound to the polyamine moleculethrough a terminal benzyl ring and/or through one or more of the aminesin the polyamine backbone. Polyamines containing aromatic rings havebeen described in the art to be structural inhibitors of the membranepolyamine transporter in mammalian cells and have been shown,themselves, not to be transported into cells. In U.S. Pat. No.5,434,145, Edwards showed bonding of alkyl-thiophosphate or alkyl-thiolgroups to one or more of the backbone amines that are present in theshort polyamine molecules. By modifying the secondary amines in thepolyamine backbone with alkyl-thiophosphate groups, the amines wereconverted to tertiary amines, and this markedly altered the basicity ofthe individual modified amine, as well as the overall polyaminemolecule. The attenuated basicity of the individual amine groups wasaccompanied by an alteration in 3-dimensional structure at these sites.With added alkyl functionality on the amine nitrogen atoms, stericbulkiness increased, so the ability or freedom of the molecule to rotateand twist at these sites was markedly reduced. The altered basicity andsteric constraints in these short spermine-like polyamines was surmisedto perturb DNA binding by the polyamine as compared to their naturalpolyamine counterparts. Consistent with this (DNA binding is abiological activity of natural polyamines), Edwards provided noinformation regarding biological activity for any of the structuresproposed in U.S. Pat. No. 5,217,964 or U.S. Pat. No. 5,434,145.

[0012] There is a need in the art, then, to create polyamine-basedmolecules that are optimized to achieve: i) local and transient growthregulation, ii) disruption of normal helical DNA structure upon binding,and iii) delivery and display of nucleophilic or other functionalmoieties within cells to enable scavenging of reactive electrophiles andradicals. There would be great advantage in developing polyaminederivatives that could be used topically to prevent or diminish thetoxic side effects of cancer chemotherapy and radiotherapy.

SUMMARY OF THE INVENTION

[0013] The present invention provides novel polyamine compounds andpharmaceutical compositions for reducing or preventing toxic sideeffects of radiotherapy and cancer chemotherapeutic agents. Thepolyamine compounds of the invention are referred to herein as“chemoprotective polyamines.”

[0014] One aspect of the invention features a compound of Formula I:

[0015] wherein:

[0016] each Z is independently A or R¹;

[0017] each A is independently:

[0018] J is a single bond or —CH(Y)—;

[0019] X is D or —R²-D;

[0020] Y is H, alkyl, or R³-D;

[0021] D is —OH, —SH, —SR⁴, or —NR⁴R⁵;

[0022] each R¹ is independently C₃₋₈ alkylene;

[0023] each R², R³, R⁶, and R⁷ is independently C₁₋₆ alkylene;

[0024] R⁴ is H or lower alkyl;

[0025] R⁵ is H, lower alkyl, or —R⁶-D;

[0026] Q is H, lower alkyl, or —R⁷—SR⁴;

[0027] k is an integer from 2 to about 16;

[0028] or a stereoisomer, prodrug, pharmaceutically acceptable salt, ormono or polyprotonated acid salt thereof.

[0029] In one embodiment, each A is independently:

[0030] In this embodiment, Y may be H or R³-D. X may be D or R²-D. Inthis embodiment, k is an integer from 2 to about 16. In specificembodiments, k is 2, 3, 4, 5, 6, 7 or 8. In other specific embodiments,k is 2-8, each R¹ is butylene, X is D, D is —NR⁴R⁵, R⁴ is H, and R⁵ isethyl, and Q is ethyl. In yet other specific embodiments, k is 2, 4, 6or 8, each R¹ is butylene, X is D, D is —SH, and Q is ethyl. Yet anotherspecific embodiment comprises a compound wherein k is 4, each R¹ isbutylene, X is D, D is —NR⁴R⁵, R⁴ is H, R⁵ is methyl, and Q is ethyl. Inother embodiments, Q is H or lower alkyl. Exemplary compounds havingthese features are shown in FIG. 1A through FIG. 1C.

[0031] In another embodiment, each A is independently:

[0032] In this embodiment, Y may be H or R³-D. X may be D or R²-D. Inthis embodiment, k is an integer from 2 to about 16. In specificembodiments, k is 2, 3, 4, 5, 6, 7 or 8. Q may be H or lower alkyl. J isa single bond; in specific embodiments, J is —CH(Y)—. Exemplarycompounds having the aforementioned features are shown in FIG. 1D andFIG. 1E.

[0033] Another aspect of the invention features a pharmaceuticalpreparation for reducing or preventing hair loss, dermatitis, mucositisor gastrointestinal distress caused by treatment with a chemotherapeuticagent or radiation therapy, which comprises at least one compound ofFormula I as described above, and a topical delivery vehicle for locallydelivering the compound to dermal or mucosal cells of skin, scalp,mouth, nasoesophageal, gastrointestinal or urogenital system. In certainembodiments, the pharmaceutical preparation further comprises at leastone other agent that reduces or prevents hair loss, dermatitis,mucositis or gastrointestinal distress caused by treatment with achemotherapeutic agent or radiation therapy, for instance, ananti-proliferative agent, a chemoprotective inducing agent or a freeradical scavenger.

[0034] The topical delivery vehicle comprises one or more of liposomes,lipid droplet emulsions, oils, aqueous emulsions of polyoxyethyleneethers, aqueous alcohol mixtures, aqueous ethanol mixtures containingpropylene glycol, aqueous ethanol mixtures containing phosphatidylcholine, lysophosphatidyl choline and triglycerides, xanthan gum inaqueous buffer, hydroxypropymethylcellulose in aqueous buffer or aqueousalcohol mixtures, diethylene glycol monoethyl ether in aqueous buffer,and biodegradable microparticles.

[0035] In a specific embodiment, the pharmaceutical preparation isformulated for topical delivery to skin or hair follicles, and thedelivery vehicle comprises an aqueous alcohol mixture and, optionally,propylene glycol. Preparations of this type may be formulated as creams,lotions, ointments or gels. In another specific embodiment, thepharmaceutical preparation is formulated for topical delivery to theoral cavity or naso-esophageal passages. In this embodiment the deliveryvehicle preferably comprises a mucoadhesive substance. It may beformulated as an aerosol, oral rinse, ointment or gel. In yet anotherspecific embodiment, the pharmaceutical preparation is formulated forvaginal or rectal delivery and comprises a mucoadhesive substance. Thesepreparations may be formulated as creams, ointments, lotions, gels,foams or suppositories. In still another specific embodiment, thepharmaceutical preparation is formulated for topical delivery to thegastrointestinal tract and the delivery vehicle comprises one or more ofnonionic liposomes and mucoadhesive substances. Preferably, thepreparation is formulated as a liquid for coating the surface of thegastrointestinal tract.

[0036] According to another aspect of the invention, methods areprovided for reducing or preventing hair loss dermatitis, mucositis orgastrointestinal distress in a patient undergoing treatment with achemotherapeutic agent or radiation therapy. The methods compriseadministering to the patient a pharmaceutical preparation as describedabove, in an amount and for a time sufficient to reduce or prevent thehair loss, dermatitis, mucositis or gastrointestinal distress. In oneembodiment, the pharmaceutical preparation is administered beginning atleast one day, and preferably up to five or more days, prior tochemotherapy or radiation therapy. In another embodiment, thepharmaceutical preparation is administered after initiation ofchemotherapy or radiation therapy. Preferably, the pharmaceuticalpreparation is administered throughout a course of chemotherapy orradiation therapy and, in certain instances continues after terminationof a course of chemotherapy or radiation therapy.

[0037] The aforementioned methods may further comprise administering tothe patient at least one other agent that reduces or prevents hair loss,dermatitis, mucositis or gastrointestinal distress caused by treatmentwith a chemotherapeutic agent or radiation therapy. These other agentsmay include anti-proliferative agents, chemoprotective inducing agentsor free radical scavengers, for instance.

[0038] The present invention also provides a method of treating cancerthat increases a patient's tolerance to high doses of a chemotherapeuticagent or radiation therapy. The method comprises (a) administering thehigh dose of the chemotherapeutic agent or radiation therapy to thepatient; and (b) administering one or more of the above-describedpharmaceutical preparations for reducing or preventing one or more ofchemotherapy- or radiation therapy-induced hair loss, dermatitis,mucositis or gastrointestinal distress, in an amount and for a time toreduce or prevent the one or more of the chemotherapy- or radiationtherapy-induced hair loss, dermatitis, mucositis or gastrointestinaldistress, thereby increasing the patient's tolerance to the high dose ofthe chemotherapeutic agent or radiation therapy.

[0039] Other features and advantages of the present invention will beunderstood by reference to the drawings, detailed description andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1A-FIG. 1E illustrate the structures of certain of thechemoprotective polyamine molecules whose synthetic pathways areillustrated in the reaction schemes. FIG. 1A shows compounds PrC 110,111, 112 and 113, olefinic core displaying —NH—CH₂—CH₃ functional group;FIG. 1B shows compounds PrC 114, 115, 116, 117 and 118, olefinic coredisplaying —SH or —OH functional group; FIG. 1C shows compounds PrC 119,120, 121, 122 and 123, olefinic core displaying —NHCH₃, —N(CH₃)₂ or —SHfunctional group; FIG. 1D shows compounds PrC 210, 211, 212, 213 and214, aliphatic core displaying —OH, —SH, —SCH₃ or —NHCH₂CH₃ functionalgroup; FIG. 1E shows compounds PrC 215, 216, 217 and 218, aliphatic coredisplaying —OH, —SH, —SCH₃ or —SCH₂CH₂N(CH₃)₂ functional group.

[0041]FIG. 2 illustrates the relationship between the number ofaliphatic carbon atoms in each chemoprotective polyamine side chain(‘arm’) and the respective IC₅₀ dose for inhibition of human fibroblastgrowth.

[0042]FIGS. 3A and 3B illustrate the level of induced p21 protein seenin diploid human fibroblasts after a 30 hr exposure to each of theindicated chemoprotective polyamines. FIG. 3B shows that the induced p21level is greater after a 30 hr exposure compared to a 50 hr exposure todrug. In these experiments, the 23SK human skin cells were exposed for30 hr to an “IC80” dose of each of the indicated chemoprotectivepolyamines and then lysed. Cell extracts were then prepared in order tomeasure p21 levels by western analysis (FIG. 3A).

[0043]FIG. 4 illustrates the relationship between the number ofaliphatic carbon atoms in each chemoprotective polyamine ‘arm’ and therespective induced p21 level in diploid human fibroblasts after a 30 hrexposure. The arrow points to the value for PrC-110, which also showedexcellent efficacy in the in vivo alopecia test.

[0044]FIGS. 5A-5D are cell histograms showing the results from flowcytometry analysis of chemoprotective polyamine-treated 23SK skin cells.FIG. 5A shows results from untreated, exponentially growing 23SK cells.FIG. 5B shows, as a control treatment, results from incubation of cellsin serum-free medium. FIG. 5C shows results from cells treated withPrC-117 for 72 hr. FIG. 5D shows results from cells treated with PrC-117for 72 hr, then switched for 48 hr to medium devoid of the PrC-117molecule.

[0045]FIGS. 6A-6E illustrate the efficacy of topically-appliedchemoprotective polyamines in protecting against chemotherapy-inducedalopecia (hair loss) in a rodent model.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0046] The present invention provides compounds for use inpharmaceutical preparations and methods for protecting non-cancerous,rapidly dividing cells in a patient's body from the toxic effects ofchemotherapeutic agents or radiotherapy administered to the patient. Inparticular, the compositions and methods of the invention are designedfor protecting epithelial cells. Most particularly, the targets areepithelial cells lining hair follicles and epithelial and/or mucosalcells of the skin, mouth, gastrointestinal (GI) and urogenital tract. Inone embodiment, the compositions are used to reduce or prevent alopeciaduring cancer therapy, by topically applying the composition to thescalp. Another embodiment comprises reduction or prevention ofgastrointestinal distress due to cancer therapy by administering thecompositions orally. Another embodiment involves reducing or preventingmucositis from chemotherapy or radiotherapy by administering thecompositions topically to the appropriate region of the body. In yetanother embodiment, the compositions are used to preventradiation-induced dermatitis, skin rash, and ulceration at the site ofirradiation by applying them to the skin.

[0047] The chemotherapeutic agents from which protection of normal cellsis desired may be one or a combination of agents used for such purpose,such as alkylating agents, antimetabolite inhibitors of DNA synthesis,antitumor antibiotics, mitotic spindle poisons, vinca alkaloids, andtopisomerase inhibitors. Specific chemotherapeutic agents include, butare not limited to, altretamine, asparaginase, bleomycin, busulfan,carboplatin, cisplatin, carmustine, chlorambucil, cladribine,cyclophosphamide (cytoxan), cytarabine, dacarbazine, dactinomycin,daunorubicin, doxorubicin, etoposide, floxuridine, fludarabinephosphate, fluorouracil, hydroxyurea, idarubicin, ifosfamide, lomustine,mechlorethamine, nitrogen mustard, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, paclitaxel, pentostatin,pliamycin, procarbazine, streptozocin, teniposide, thioguanine,thiotepa, vinblastine and vincristine. The radiation therapy consists ofall useful types of radiation used in cancer treatment, includingx-rays, gamma-rays, electron beams, photons, alpha-particles andneutrons.

[0048] Commonly-owned, co-pending U.S. application Ser. No. 10/214,917and International Application No. PCT/US02/25216, each filed Aug. 7,2002, describe that several types of known polyamines and polyamineanalogs, referred to therein as “polyamine effector” compounds, can beefficiently delivered to the aforementioned target cell populations,where they are capable of protecting those cells from the harmful sideeffects of chemotherapy or radiotherapy. The present invention providesnovel polyamine compounds specifically designed for improved efficacy inprotecting normal cells from the detrimental effect of cancerchemotherapy or radiation therapy. These molecules are referred toherein as “chemoprotective polyamines.”

[0049] Certain definitions that will assist in the understanding of thepresent invention are set forth below, while others are providedthroughout the specification. With respect to the compounds of theinvention, it should be noted that if any variable occurs more than onetime in any constituent or in any formula, its definition in eachoccurrence is independent of its definition at every other occurrence.Thus, for example, if a compound of the present invention is shown toincorporate, for example, one or more of R¹, R², R³, R⁴, R⁵, R⁶, R⁷,—OH, —SH, —SR⁴, or —NR⁴R⁵, then the R¹, R², R³, R⁴, R⁵, R⁶, R⁷, —OH,—SH, —SR⁴, or —NR⁴R⁵ at each occurrence is selected independently.Combinations of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, —OH, —SH, —SR⁴, or —NR⁴R⁵are permissible only if such combinations result in stable compounds.

[0050] Polyamines are small aliphatic amines found in all living cells.By nature, polyamines within cells are polycationic (i.e., capable ofsustaining or neutralizing one or more equivalents of acid). They arebiosynthesized from amino acids, such as arginine and ornithine.Examples of common polyamines found in plant and animal cells are:putrescine (NH₂(CH₂)₃NH₂), formed by the decarboxylation of ornithine orarginine; spermidine (NH₂(CH₂)₃NH(CH₂)₄NH₂); and spermine(NH₂(CH₂)₃NH(CH₂)₄HN(CH2)₃NH₂); the latter two being formed bysubsequent addition of an aminopropyl moiety to putrescine andspermidine, respectively. Because such polyamines are found in nature,they may be referred to as “naturally-occurring” polyamines. However,they may be prepared by a variety of synthetic strategies, as would beknown in the chemical arts.

[0051] The term “polyamine analogs” as used herein refers topolycationic molecules that are similar, but not identical to polyaminesfound in nature. Polyamine analogs may be branched or unbranched, or mayhave other structural variations as compared to naturally-occurringpolyamines, while retaining the central features of polyamines (multipleamine groups, polycationic within cells). Polyamine analogs may befurther categorized into three groups: (1) simple polyamine analogs, (2)constrained or conformationally restricted polyamine analogs, and (3)linked or long-chain polyamine analogs.

[0052] A “simple polyamine analog” retains the flexibility conferred bythe aliphatic carbon backbone, as well as the approximate carbon chainlength of naturally-occurring polyamines, but possess a modification orcontain one or more added functional groups (e.g., sulfhydryl, phenyl,alkyl) that confers a desired feature or advantage to the molecule.

[0053] By comparison, “conformationally restricted polyamine analogs”(sometimes referred to herein as “constrained polyamine analogs” aremodified in their carbon backbone to remove flexibility in the modifiedarea, such that two or more amino functionalities in the molecule arerestricted to a particular spatial location. Such modification often isaccomplished by introducing a cyclic or unsaturated moiety at one ormore locations in the carbon backbone, as described in greater detailherein.

[0054] “Linked or long-chain polyamine analogs” are polyamines that arelonger than naturally-occurring polyamines such as spermine. Increasingthe overall length of a polyamine may be accomplished, for example, bylinking together oligoamines or by adding oligoamine “units” (such asaminopropyl or aminobutyl groups) to a foundation molecule, such asspermine. Thus, while spermine has a 3-4-3 carbon backbone (4 carbonsbetween the two internal amino groups and 3 carbons between eachinternal amino group and the respective terminal amino groups), linkedor long-chain analogs might comprise an additional one, two, three, fouror more aminopropyl or aminobutyl groups, for example, on either or bothends of the molecule, and further may comprise terminal methyl or ethylgroups on either or both ends.

[0055] As used herein, the term “antiproliferative” refers to an agentthat slows or stops cell division. The antiproliferative agent may exertits effect by inhibiting cell cycle progression at one or more stages.Such an agent may be referred to herein as a “cell cycle progressioninhibitor.” The chemoprotective polyamines of the invention can act asantiproliferatives, specifically cell cycle progression inhibitors, byassociating with and modifying the conformation or structure of DNA.These agents are sometimes referred to herein as “DNA modifiers.”

[0056] The design of the chemoprotective polyamines of the presentinvention emerges from the inventors' appreciation of the advantagesassociated with blending certain important chemical properties within asingle multifunctional molecule, 1) molecular structure necessary forefficient binding to DNA and, in some instances, modification of theconformation or structure of DNA; 2) nucleophilic reactivity, to trapelectrophilic chemicals that can challenge the integrity of helical DNA;and/or 3) free radical-scavenging activity to reduce or eliminate freeradicals often generated by irradiation or various chemotherapeuticagents (e.g., certain reactive oxygen species).

[0057] In regard to structure, the ability of a polyamine to physicallyalign closely, or “dock” with DNA should be maintained. Mimicking thegeneral linear nature of the known natural polyamines enables thechemoprotective polyamines of the invention to maintain DNA bindingability. Another important feature common to natural polyamines is thepresence of multiple secondary amine nitrogen atoms throughout thebackbone. These atoms are known to be protonated, and thus positivelycharged, at physiologic pH. Accordingly, maintaining secondary aminefunctionality throughout a chemoprotective polyamine further providessufficient active binding sites.

[0058] Nucleophilic and/or free radical-scavenging activity was designedinto the chemoprotective polyamines with the aim of maintaining all ofthe above mentioned structural and binding features. In variousexemplary embodiments described herein, electron-rich groups, bearingsp3-hybridized nitrogen, sulfur or oxygen atoms, were positionedstrategically within the polyamine backbone so that overall linearityand secondary amine character would be preserved for efficient DNAbinding. The enhanced reactivity of allylic functional groups, comparedto their alkyl counterparts, was also considered in designing placementof functional groups in certain embodiments. In some embodiments,chemoprotective polyamines with an olefinic core have thenucleophiles/scavengers positioned on allylic positions specifically toenhance the reactivity of those functional groups. In these embodiments,the core segment bearing the functional group was restricted in size,consistent with natural polyamine features, and provides a suitableplatform from which the nucleophile or other functional group isdisplayed. This design feature allows one side, or face, of the3-dimensional polyamine structure to interact with DNA while the otherface, bearing the reactive functional group, is projected away from theDNA, sterically unencumbered, thus free to react with toxicelectrophilic chemicals or free radicals present in the cellular matrix.

[0059] The chemoprotective polyamines of the present invention arerepresented by the general structure of Formula I:

[0060] In Formula I, “Z” is either “A” or “R¹.” “A” represents a “core”segment and the R¹ and Q groups typically represent alkylene (R¹) oralkyl (Q) chains of varying length (branched or unbranched), which,together with the amine groups as shown, make up the linked oligoaminesegments that form the polyamines of the present invention.

[0061] As used herein, “alkylene” refers to a bivalent alkyl radicalhaving the general formula —(CH₂)_(n)—, where n is 1 to about 8.Non-limiting examples include methylene, ethylene, trimethylene,butylene, pentamethylene, and hexamethylene. Alkylene groups may bebranched or unbranched. Alkylene groups may also contain one or moredouble or triple bonds within the backbone of the —(CH₂)_(n)— moiety,provided that the resultant compound is stable. Non-limiting examplesinclude —CH₂—C≡C—CH₂— and CH₂—CH═CH—CH₂—. Alkylene groups can besubstituted or unsubstituted, provided that the resultant compound isstable and so long as the substituent does not substantially interferewith present compound's intended mode of action. In certaincircumstances, alkylene is preferably C₃₋₈ alkylene, while in othercircumstances, even within the same molecule, alkylene is preferablyC₁₋₆ alkylene.

[0062] As used herein, “alkyl” refers to a saturated straight orbranched hydrocarbon having from about 1 to about 20 carbon atoms (andall combinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 1 to about 8 carbon atoms, hereinreferred to as “lower alkyl”, being preferred. Alkyl groups include, butare not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, octyl, decyl,dodecyl, octadecanyl, and eicosanyl.

[0063] The core segment (“A”) functions in two ways: (1) it presents aplatform for display of a protective functional group, namely anucleophile or a free radical scavenger; and (2) it may be designed tointroduce a conformational constraint to the polyamine (e.g., a doublebond or a cyclic structure). The linked oligoamine segments (sometimesreferred to as “arms” or as “polyamine side chains”) function to enablethe molecule to “dock” with DNA, as do naturally occurring polyamines.In one embodiment, a compound of the invention comprises one core and an“arm” of varying length on either side of the core. In anotherembodiment, the core may have a single arm (i.e., the core group is atone end or the other of the polyamine molecule). In another embodiment,the chemoprotective polyamine comprises two or more cores (which may bethe same or different), which can be side-by-side or separated by anoligoamine segment of varying length.

[0064] The core segment provides the molecule with conformationalrestraint and/or a protective functional group that is attached(“tethered”) to the molecule in such a way as to be optimally availablefor interaction with electrophilic groups, free radical groups and otherreactive species present on or generated by chemotherapeutic agents orradiation. In the present invention, conformation restraint is typicallyintroduced through the use of a double bond between two carbons. Aswould be appreciated by those of skill in the art, other means ofintroducing conformational restraint include triple bonds and ringstructures, such as three-, four-, five- and six-carbon or moresubstituted or unsubstituted rings (in the latter embodiments, with theproviso that the ring does not introduce bulk or steric hindrance thatreduces the ability of the functional group to access its targets).

[0065] The protective functional groups displayed on the core aredesigned to act as nucleophiles or as free radicalscavengers/antioxidants, with the understanding that certain functionalgroups may carry out both functions. Functional groups that typicallyact as nucleophiles, but that may also act as antioxidants or freeradical scavengers, include, but are not limited to, —OH, —NH₂, —NHR,NR₂, —SH and —SR(wherein R is methyl or a lower alkyl which itself maybe substituted with —OH, —NH₂, —NHR, NR₂, —SH or —SR).

[0066] The total length or size of a chemoprotective polyamine of theinvention is generally described herein by the number of oligoaminesegments (R¹-NH—) that make up the molecule. Typically the compoundscomprise two or more such segments, and may comprise 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 or even more such segments. The overallupper limit to length of the compounds is typically selected on thebasis of practical considerations such as cost and ease of synthesis,solubility and/or skin or mucosal permeability, as measured againstefficacy of the compound in exerting its protective effect within thecell. In specific embodiments, the chemoprotective polyamine comprises2, 3, 4, 5, 6, 7 or 8 oligoamine segments.

[0067] 1. Synthesis of Chemoprotective Polyamines ComprisingNucleophilic Cores

[0068] The synthetic approaches illustrated below demonstrateversatility regarding the choice of nucleophile incorporated into thecore segments, the availability of both cis- and trans-isomers of theolefinic core and variability in the amine side chain segment length aswell as number of segments desired. Several reaction schemes and tablesare presented throughout the sections below, with both reactionintermediates and final products being assigned unique descriptivenumbers. Specific descriptions of the synthesis of the key molecules areset forth in Example 1.

[0069] 1.1 Amine Side Chains

[0070] In exemplary embodiments of the invention, amine side chains weresynthesized using the reaction sequences in Scheme 1. Primary alkylamine 1 was converted to mesitylene sulfonamide 2, which was alkylatedto provide N-phthaloyl protected 3. It should be noted that the segmentlength can be adjusted from two carbons to six carbons in this sequenceof steps, and this invention is not limited to the four-carbon chainlength of molecule 3. Deprotection of the terminal nitrogen gave 4,which was readily converted to 5. The bis-sulfonamide 5 represents theshortest amine side chain with regard to number of segments. Molecule 5also was used for chain elongation by adding segments. The threereaction steps that convert 2 to 5 were repeated and therefore representan iterative process by which 5 was converted to 8, 8 elaborated to 11and 11 to 14. Each of the mesitylenesulfonyl protected amine side chains5, 8, 11, 14, and related chain-extended derivatives, are suitable forattachment to a core segment. In sum, Scheme 1 describes how a singlepolyamine side chain may be produced. This process can be repeated toadd additional polyamine side chain segments.

[0071] 1.2 Synthesis of Olefinic Core and Side Chain Attachment

[0072] A general description of the olefinic core synthesis isillustrated in Scheme 2. Dihydroxyacetone dimer 15 was converted toketone 16. Olefination of 16 provided ester 17, which was carefullyreduced to the allylic alcohol 18 while maintaining the integrity of thesilyl groups. Mesylation gave allylic mesylate 19, which was coupled toan amine side chain to provide 20, where A represents themesitylenesulfonyl protected amine side chain. Acid treatment of 20 gavediol 21, which was monobenzoylated to provide 22. It should be notedthat the cis- and trans-isomers of alcohol 22 can be separated bychromatography to provide the individual purified isomers. Alcohol 22was then transformed to the allylic bromide 23, which was coupled to asecond protected amine side chain to produce 24. For the purpose of thisinvention it should be noted that in 24, protected amine side chains Aand A′ can be identical, but can also vary in segment length as well asoverall chain length. Hydrolysis of 24 gave mesitylenesulfonyl protectedpolyamine 25. Protected polyamine 25 can be deprotected (see polyamine27 in Scheme 3), or serve as a versatile intermediate that can befurther elaborated at the allylic alcohol position to insert alternativeprotective functional groups.

[0073] 1.3 Functional Groups on the Chemoprotective Polyamine Core

[0074] A method for introducing various protective functional groupsonto a core segment is shown in Scheme 3. Alcohol 26 was converted tomesylate 27, which was subsequently reacted with various species havingsuitable nucleophilic character, to provide, for example, 29, 31, 33 or35. Subsequent deprotection produced, for example, the chemoprotectivepolyamines 30, 32, 34 and 36.

[0075] 1.4 Functional Groups Displayed from an Aliphatic Core

[0076] A synthetic approach to chemoprotective polyamines bearingfunctional groups on an aliphatic core segment is shown in Scheme 4.

[0077] In some pharmacologic settings, there may be advantage indisplaying a protective functional group from a flexible aliphatic coreas has been done in molecules PrC-210, PrC-211, as well as the rest ofthe molecules shown in FIG. 1D and 1E. Using chemoprotective polyaminesto deliver nucleophiles/free radical scavengers to at-risk cells, whilealso binding DNA to enable DNA protection and growth regulation,requires optimization of each of the chemoprotective polyamine'sstructural parameters, including segment length, overall length,functional group, and the platform from which the functional group isdisplayed. For instance, displaying an alkyl-nucleophile side chain froma flexible core may change the interaction between polyamine and DNA,and with it, change the growth regulation “phenotype” that would belinked with the displayed nucleophile “phenotype” on a particularchemoprotective polyamine. This combination of functions within a givenmolecule may be optimized for each pharmacologic use of chemoprotectivepolyamines. In the reaction sequence of Scheme 4, dichloride 37 wasconverted to olefin 38, which was subsequently transformed to alcohol39. Alcohol 39 can be deprotected to give 40, or converted to themesylate intermediate 41. Mesylate 41 was then converted, with suitablenucleophiles, to 42 and 44, which upon deprotection, producechemoprotective polyamines 43 and 45.

[0078] Other aliphatic polyamines may be prepared by hydrogenatingolefinic polyamines of the invention. This is accomplished by employinghydrogenation catalysts in the presence of hydrogen or molecules thatprovide hydrogen during the course of a reaction, such as for example,hydrazine, cyclohexadiene, or alpha-terpinene. Further, as oneordinarily skilled in the art would recognize, one or more of the doublebonds in any given olefinic polyamine may be selectively hydrogenated byselection of catalysts that preferably coordinate to one or more of the“D” moieties of the present compounds and transfer hydrogen selectivelyto the olefin adjacent to the “D” moiety. For a general overview, see J.March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,Fourth Edition, John Wiley and Sons, New York (1992), pp 771-780.

[0079] 1.5 Polyamines Containing Two or More Cores

[0080] The synthesis of a chemoprotective polyamine with more than onecore unit is illustrated in Scheme 5. The core intermediate 23 (seeScheme 2) is reacted with sulfonamide 54 to give 55. Removal of thephthaloyl group provides 56, which upon sufonylation givesbis-sulfonamide 57. If the desired functional group on the core segmentsof the target polyamine is hydroxyl, 57 can be converted directly tosilyl ether 60, where X═OH. Alternatively, the nucleophile can bemodified by converting 57 to 58, followed by functional grouptransformation to 59. Sulfonamide 59 can likewise be converted to 60.Desilylation to 61, and subsequent benzoylation, gives 62. Conversion tothe bromide 63 provides a pivitol intermediate. The chain-elongationprocess can be terminated by attaching an amine side chain, thusproviding a polyamine bearing two core units. Alternatively, bromide 63can be subjected to an iterative process that involves repeating thesteps shown at the start of Scheme 5. This will install a thirdlinker-core repeating unit in the polyamine chain. Manipulation of thefunctional group in a second or third core unit can be effected as shownin the conversion of 57 to 59.

[0081] 2. Utility of Chemoprotective Polyamines in Regulating CellGrowth and Protecting Against Cancer Therapies

[0082] To determine the activity of the described compounds asregulators of cell growth, as well as to provide mechanistic insightinto ways by which these compounds regulate cell growth, we examinedchemical interaction between the subject compounds and nucleic acids andassessed the extent to which these chemical and growth regulatoryproperties conferred protection in animal tissues against cancerchemotherapy and radiotherapy. Several exemplary compounds of theinvention were tested using various in vitro and in vivo model systems.The subject compounds were found to inhibit growth of human skin cellsat sub-micromolar to millimolar concentrations, in a manner that couldbe correlated to their chemical structure. Consistent with thisinhibition of cell growth, the compounds were shown to bind avidly tohelical DNA, to induce expression of the negative growth regulator, p21,and to block cells within the G1 phase of the cell cycle, also in amanner related to their structure. When the subject molecules wereapplied locally by topical administration to rodent skin, they protectedthe hair follicle cells and blocked the alopecia normally seen followingsystemic administration of a chemotherapy drug.

[0083] The in vitro growth inhibitory effects of certain of thechemoprotective polyamines of the invention were measured using primary,diploid fibroblasts isolated from human skin. As shown in Table 1, IC₅₀concentrations (the drug concentration that caused a 50% inhibition ofcell growth) for the polyamines ranged from sub-micromolar tomillimolar. TABLE 1 Expt. 1, Expt. 2 Compound # [MW:HCl salt] IC₅₀ (uM)PrC-110 [523.9] 1680 809 PrC-111 [739.0] 980 180 PrC-112 [954.2] 2.53PrC-113 [1169.3] 0.33 0.21 PrC-114 [476.4] 850 240 PrC-115 [691.61 41001090 PrC-116 [906.7] 5.8 PrC-117 [1121.9] 0.32 0.24 PrC-118 [675.5] 78PrC-119 [725.0] 470 202 PrC-120 [1155.3] 0.22 0.18 PrC-121 [1169.3] 0.15PrC-122 [940.2] 0.81

[0084] As part of this invention, heretofore not described in theliterature, FIG. 2 shows that the IC₅₀ concentration for eachchemoprotective polyamine was tightly correlated with the length of thepolyamine side chains (‘arms’) attached to a central butene core, withthe long arms, i.e., those containing 16 aliphatic carbon atoms,associated with sub-micromolar IC₅₀ values.

[0085] The chemoprotective polyamines of the invention are also able tobind, denature and precipitate DNA from solution. As is known in thefield, as the concentration of polyamine is increased, there is a pointwhere polyamine binding to helical B-DNA induces single-stranded‘bubbles’ and conversion to other forms of DNA structure, such as Z-DNA(Feuerstein, B. et al. Nuc. Acids Res. 17:6883-6892, 1989; Basu, H. andMarton, L. Biochem. J. 244:243-246, 1987), as well as precipitating theDNA from solution. Table 2 shows that the four molecules that contain‘16 carbon arms,’ i.e., PrC-113, PrC-117, PrC-120 and PrC-121, all haveIC₅₀ concentrations that are lower than all of the other molecules thatcontain shorter aliphatic arms. TABLE 2 DNA Bind/ppt. Expt. 1, Expt. 2Compound # [MW:HCl salt] IC₅₀ (uM) PrC-110 [523.9] 270  PrC-111 [739.0]88 PrC-112 [954.2] 93 PrC-113 [1169.3] 35 PrC-114 [476.4] 94 PrC-115[691.6]  37, 83 PrC-116 [906.7]  82, 63 PrC-117 [1121.9]  58, 57 PrC-118[675.5] — PrC-119 [725.0] 89 PrC-120 [1155.3] 57 PrC-121 [1169.3] 49PrC-122 [940.2] 102  PrC-123 [906.7] 131,  117  Spermine 2600 

[0086] This relationship between arm length of the chemoprotectivepolyamine and increased ability to disrupt and denature B-DNA structureis also a unique aspect of this invention. The increased ability to bindDNA and disrupt its helical structure may also contribute to themolecule's ability to protect cellular DNA against electrophilicchemotherapy drug metabolites and against oxygen free radicals generatedduring radiotherapy. Both of these toxic modalities are believed torequire normal B-DNA helical structure within the cell's nuclear DNA inorder to achieve chemical or physical disruption of the cellular DNA,the first step in the apoptotic cascade.

[0087]FIG. 3 illustrates that chemoprotective polyamines are able toinduce expression of the negative cell cycle regulatory protein, p21,after exposing the human skin cells to the polyamine molecules. FIG. 3Bshows that the induced p21 level is greater after a 30 hr exposurecompared to a 50 hr exposure to drug. In these experiments, the 23SKhuman skin cells were exposed for 30 hr to an “IC₈₀” dose of each of theindicated chemoprotective polyamines and then lysed. Cell extracts werethen prepared in order to measure p21 levels by western analysis (FIG.3A). Results are summarized in Table 3. Although the ability of modifiedpolyamines to induce p21 is known in the literature (Kramer, D. et al.,Cancer Res. 59:1278-1286, 1999), it is a novel aspect of this inventionthat those chemoprotective polyamines with longer aliphatic “arms” werebetter able to induce expression of p21 as shown in FIG. 4. TABLE 3 p21fold-induction Compound # [MW:HCl salt] at IC₈₀ Dose PrC-110 [523.9]2.91 PrC-111 [739.0] 2.22 PrC-112 [954.2] 2.45 PrC-113 [1169.3] 3.21PrC-114 [476.4] — PrC-115 [691.6] — PrC-116 [906.7] 2.32 PrC-117[1121.9] ˜3.0  PrC-118 [675.5] — PrC-119 [725.0] 1.80 PrC-120 [1155.3]2.01 PrC-121 [1169.3] 3.26 PrC-122 [940.2] 1.70 PrC-123 [906.7] 2.22Colcemid 3.31

[0088] In FIG. 5, cell histograms showing the results from flowcytometry analysis of chemoprotective polyamine-treated 23SK skin cellsare shown. FIG. 5A shows that for untreated, exponentially growing 23SKcells, 59.12% of the cells are present in the S+G2 cell cyclecompartments, whereas only 40.88% of the cells are in the G1compartment. FIG. 5B shows, as a control treatment, that incubation ofcells in serum-free medium causes a sizable reduction in S+G2 cellcompartments (down to 5.63% total), and a sizable increase in cells nowpresent in the G1 compartment (up to 94.37%). FIG. 5C shows that cellstreated with PrC-117 for 72 hr also show a marked reduction in S+G2compartments (down to 13.77%) and a marked increase in the G1compartment (up to 86.23%). FIG. 5D shows that after the cells treatedwith PrC-117 for 72 hr are switched for 48 hr to medium devoid of thePrc-117 molecule, the distribution within cell cycle compartments isbasically returned to that seen in cells previously untreated withchemoprotective polyamine (i.e., FIG. 5A). The transient nature of thecell cycle block induced by chemoprotective polyamines is believed to bean important aspect of their efficacy, i.e., their ability to block cellcycle progression in stem cells during the course of chemo- orradiotherapy, and the resumption of normal stem cell division after agiven cancer therapy course has been completed. Table 4 shows that, ofthe nine chemoprotective polyamine molecules tested, three caused G1cell cycle blocks with greater than 75% of the cells present in the G1compartment, and each of these three molecules contained 16 carbonaliphatic arms. TABLE 4 Cell Cycle Distribution At IC₈₀ Dose (%)Compound # [MW:HCl salt] G1 S G2/M PrC-110 [523.9] 60 26 14 PrC-111[739.0] 60 25 14 PrC-112 [954.2] 61 23 16 PrC-113 [1169.3] 77 8 14PrC-114 [476.4] — PrC-115 [691.6] — PrC-116 [906.7] 66 11 23 PrC-117[1121.9] 86 11  3 PrC-118 [675.5] — PrC-119 [725.0] — PrC-120 [1155.3] —PrC-121 [1169.3] 76  4 20 PrC-122 [940.2] 67 15 18 PrC-123 [906.7] 68 1021 Colcemid  8 11 81

[0089] Natural polyamines such as spermine, with a 3-4-3 configurationof aliphatic carbon chains containing terminal amine groups andseparated by intervening amine groups, are known to bind avidly tocellular DNA in the cell setting. Synthetic polyamines, containinglonger aliphatic carbon segments, typically of four carbons, have beenshown to displace natural polyamines like spermine from DNA because oftheir greater binding affinity for helical DNA. At physiologic pH, eachof the amine groups of a polyamine backbone can protonated to yield anammonium cation. Therefore, as the length of a polyamine increases,achieved by oligomerizing a —(CH₂)₄—NH— segment, for example, there aretypically an increased number of ammonium cations distributed along thepolyamine backbone for bonding with anions distributed along the DNAbackbone. As a result, longer, synthetic polyamine analogs compete moreeffectively with spermine in vitro and in vivo for binding to DNA.Binding of polyamines to helical DNA has also been shown to conferconformational changes to the DNA, such as conversion of helical B DNAto A or Z forms of DNA. And, in vivo, polyamine analogs have also beenshown to cause condensation and aggregation of DNA and chromatin withinmammalian cells (Basu, H., et al., Cancer Res. 49: 5591, 1989; Basu, H.et al., Biochem. J. 269:329, 1990). Though not intending to be bound byany particular theory, it is believed that his tight binding andassociated distortion of normal helical structure, which is optimized inthe design of chemoprotective polyamines of the present invention,provides pharmacologic benefit in at least three ways. First, thepharmacologic, growth inhibitory activity is reversible, as shown, forexample, for the PrC-117 molecule in FIG. 5, i.e., by simply stoppingtopical application, the treated cells are released from growthinhibition thus yielding a ‘transient’ growth regulation. Second,distortion of helical DNA and the formation of single-stranded bubblesis likely to be the cause, or to be closely related to the cause, of theinduced expression of p21 and the G1 cell cycle block that is associatedwith its induced expression. Third, for many electrophilic, alkylatingdrugs, reaction with DNA occurs in two steps, the first step requiringintercalation of the drug molecule between nucleoside bases in helical BDNA, and a rapid second step involving alkylation of the adjacent DNAbase by the drug molecule. By condensing and altering normal DNA helicalform, chemoprotective polyamines are expected to significantly reducealkylation of cellular DNA by electrophilic drugs. Likewise,condensation and alteration of DNA helical form by polyamine binding invitro has also been shown to dramatically reduce the number of singlestrand breaks induced when the DNA is directly irradiated in vitro(Spotheim, M., Int. J. Radiat. Biol. 68: 571-577, 1995).

[0090] When comparing chemoprotective polyamines to those polyamineanalogs that have been previously described, there are a number ofmarked differences. For instance, Edwards (U.S. Pat. Nos. 5,217,964 and5,434,145) attached one or more alkyl-thiophosphate or alkyl-thiolgroups to one or more of the backbone amines of short aliphaticpolyamines, or to one or more backbone amines as well as to one or moreterminal benzyl groups on equally short polyamines. In comparison, thepresent inventors have designed and synthesized chemoprotectivepolyamine molecules which: i) optimize both the polyamine side chain(“arm”) length and overall molecule length to achieve tight DNA binding,ii) project or “display” a protective functional group physically awayfrom the DNA to which the chemoprotective polyamine is strongly bound,iii) attach the functional group to a polyamine backbone carbon atominstead of to one of the backbone amine groups, iv) in certainembodiments, display functional groups from allylic positions ofolefinic core segments that are present in chemoprotective polyamines;this is done by design to enhance reactivity of the group, ν) include arange of functional groups that are “displayed,” including —SH, —OH,—NH₂, —NHR, —NR₂, —SH and —SCH₃ moieties, singly or in combination, aswell as other groups that are known to vary in their degree ofnucleophilicity or ability to scavenge free radicals, vi) include thedisplay of more than one functional group per polyamine molecule, andvii) in some embodiments, include a rigid platform from which thefunctional group is projected or displayed on a spacer aliphatic chainaway from the DNA in a manner that better enables the “sentinel group”to scavenge or trap electrophiles/oxygen radicals from the cellularmilieu before they attack other known nucleophilic groups within DNA,such as the 2-amino group of deoxyguanosine.

[0091] This ability to scavenge and trap chemical/physical reactantswithin a cell does not require the chemoprotective polyamine to bephysically attached to cellular DNA or RNA. Rather, simple molarpresence of such nucleophilic or other protective functional groups incells would be expected to be protective. For instance, previous work inthe field has shown a positive, linear correlation between theintracellular concentration of the physiologic nucleophile, glutathione(GSH), and the concentration of an electrophile required to kill theexposed cells (Ho, D. and Fahl, W. E., J. Biol. Chem. 259: 11231-11235,1984). In another mechanism by which chemoprotective polyamines mightprotect cells against cytotoxic threats, they may serve as a “stealth”vehicle by which to load cells with —SH or other nucleophilic orprotective groups. Whereas, it is well known in the field (Levy, E. etal., Proc. Natl. Acad. Sci. USA 90:9171-9175, 1993) that theSH-containing nucleophile, glutathione, is not taken up by cells in aphysiologic setting, the cell membrane polyamine transporter (PTS),which is known to mediate the uptake of polyamines, molecules containingmultiple charged sites, should efficiently transport functionalgroup-displaying polyamines into cells, and that this would provide anefficient means to “load” cells with, e.g., an SH-containing polyamine,which could serve as a glutathione surrogate. Once loaded with thepolyamine, these cells would be protected from subsequent toxicchallenges, such as those seen with transient chemotherapy andradiotherapy regimens. The results in the tables and figures that showthe same growth regulating efficacy for each of the SH-containingchemoprotective polyamines (i.e., PrC-114, PrC-115, PrC-116, PrC-117) asfor those chemoprotective polyamines without SH groups implies that theSH-containing molecules are transported into the human fibroblastsequally well, and that they bind with equal affinity to cellular DNA.Moreover, the fact that each of the SH-displaying chemoprotectivepolyamines exemplified herein has shown protective activity in the ratcytoxan-induced alopecia assay demonstrates that the displayednucleophile is also active within the cell milieu.

[0092] Another way to increase the molar presence ofnucleophiles/scavengers within the nuclear environs is to display morethan one such functional group on each chemoprotective polyaminemolecule. In embodiments where two —SH groups are displayed on a singlepolyamine, then a reducing agent such as sodium borohydride or others asknown in the art may added to the pharmaceutical preparation to reduceany —S—S— disulfide bonds that might be formed when —SH groups arepresent in an oxygen containing medium. An alternate strategy to avoiddisulfide bond formation is to “cap” the displayed sulfur atom with aCH₃ group to prevent interaction of the sulfur atoms, while stillretaining the capacity of the sulfur atom to scavengeelectrophiles/oxygen radicals.

[0093] The use and placement of protective functional groups on thebackbone of chemoprotective polyamines is also significantly differentfrom the attachment of —CH₂CH₂SPO₃H₂ or —CH₂CH₂SH groups to polyaminesdescribed by Edwards in U.S. Pat. Nos. 5,434,145 and 5,217,964. In U.S.Pat. No. 5,434,145, Edwards showed bonding of alkyl-thiophosphate oralkyl-thiol groups to one or more of the 3-4 backbone amines present inthe short polyamine molecules. By modifying the secondary amines in thepolyamine backbone with alkyl-thiophosphate groups, the amines wereconverted to tertiary amines, which markedly alters the basicity of theindividual modified amine, as well as the overall polyamine molecule.The attenuated basicity of the individual amine groups is accompanied byan alteration in 3-dimensional structure at these sites. With addedalkyl functionality on the amine nitrogen atoms, steric bulkinessincreases, so the ability or freedom of the molecule to rotate and twistat these sites is markedly reduced. The altered basicity and stericconstraints in these short spermine-like polyamines perturbs DNA bindingby the polyamine as compared to their natural polyamine counterparts.Given the already very high IC₅₀ concentration of spermine for DNAbinding/precipitation (nearly 1,000-fold higher than for mostchemoprotective polyamines; see Table 2), it is possible that themodification of backbone amines described by Edwards would eliminate DNAbinding altogether in cells at the concentrations of drug that could bepharmacologically achieved. The attenuated basicity of theamine-modified polyamine molecules in Edwards could also affect theirpharmacologic delivery characteristics. In topical applications to skinand other epithelial surfaces, there is an accepted relationship betweenthe degree of ionization at physiologic pH of an applied drug and thedegree to which it permeates or traverses the surface cells. In contrastto Edwards, the functional group used in the chemoprotective polyaminesof the invention, whether —SH or one of several other groups (e.g., OH,N-ethyl, N-methyl, N-dimethyl; see FIG. 1), is bound to a carbon atomwithin the polyamine backbone. This was done specifically to avoidperturbing the DNA binding characteristics of each of the backbone aminegroups, while still achieving the display of reactive functional groups.

[0094] In U.S. Pat. No. 5,217,964, Edwards discloses the linking of oneor more alkyl-thiophosphate or alkyl-thiol groups to the polyaminebackbone through one or more terminal benzyl group(s) or through one ormore of the backbone amine groups. Work within the field (Huber, M., J.Biol. Chem. 271:27556-27563, 1996) has shown that polyamines containingone or more aromatic groups are well-suited to serve as inhibitors ofthe membrane polyamine uptake transporter, and predictably, theythemselves are not taken up into cells. Consistent with the aboveobservations, Edwards provides no information regarding biologicalactivity for any of the structures proposed in U.S. Pat. No. 5,217,964or U.S. Pat. No. 5,434,145.

[0095]FIGS. 6A-6E illustrate the efficacy of each of the indicatedchemoprotective polyamines in protecting against Cytoxan-inducedalopecia in the rat model (Hussein et al., 1990, infra). In thisprotocol (See Example 2), chemoprotective polyamines are appliedtopically to the rat pups' backs in an alcohol:water delivery vehicle,once per day, for five days before and five days after a single systemicdose of Cytoxan. As seen, topical chemoprotective polyamines conferredsignificant protection against the generalized alopecia that was seen tooccur in the vehicle-treated rat pups.

[0096] 3. Topical or Local Administration of Pharmaceutical Preparations

[0097] As described above, the chemoprotective polyamines of the presentinvention have been shown to inhibit the growth of normal human skincells, to modify normal B-DNA helical structure, to induce expression ofthe negative cell cycle regulator, p21, to cause a G1-specific cellcycle block, and to protect against chemotherapy-induced alopecia anddermatitis in an animal model. Thus, the compounds of the invention areparticularly suitable for treatment of humans to prevent the local sideeffects of cancer chemotherapy and radiotherapy. Based upon their growthregulatory effects, chemoprotective polyamines may also find utility inother applications where inhibition of cell growth would beadvantageous, including regulating proliferative conditions of the skin,such as psoriasis and dermal nevus.

[0098] Two important targets for delivery of such protective therapiesare (1) the epithelial cells of the skin, including hair follicles andthe epidermis, and (2) the epithelial cells lining the oral and entiregastrointestinal (GI) or urogenital tract. The method of protection ofthese tissues with chemoprotective polyamine comprises administering toa population of epithelial cells a composition consisting of achemoprotective polyamine and a delivery vehicle for a time and in anamount effective to protect the non-neoplastic cells from damage duringthe cancer chemotherapy or radiotherapy. In one embodiment, the methodis used to prevent alopecia during cancer therapy, by topically applyingthe composition to the scalp. In another embodiment, the method is usedto prevent gastrointestinal distress due to cancer therapy byadministering the composition orally. In another embodiment, the methodis used to prevent mucositis from chemotherapy or radiotherapy byadministering the composition topically to the appropriate region of thebody. In yet another embodiment, the method is used to preventradiation-induced dermatitis, skin rash, and ulceration at the site ofirradiation by applying the composition to the skin.

[0099] Administration of chemoprotective polyamines to human ornon-human subjects can be achieved in several ways. The preferredadministration route is topical, to tissue sites including the skin, aswell as oropharyngeal and gastrointestinal mucosal surfaces. It can alsobe delivered locally to an internal organ, tissue or regions thereof. Itshould be noted, as with all pharmaceuticals, the concentration andtotal amount of polyamine administered will vary depending upon thetissue being treated, the mode of administration, the size and conditionof the subject being treated, and the particular chemoprotectivepolyamine being used.

[0100] Compositions of chemoprotective polyamines formulated in deliveryvehicles are well-suited to be administered topically to the skin orsurfaces of the mouth, GI or urogenital tract. Pharmacologicconcentrations of chemoprotective polyamines can protect normal,non-neoplastic cells from cancer therapy-associated cell damage. Byproducing a local gradient effect within the tissues, the topicallyapplied polyamine produces a local protective effect at the intendedregion. This dose-dependant gradient of topical drug can effectivelyprotect normal proliferating cells rendering them less susceptible toradiation or chemotherapy. Importantly, while this local effect wouldprotect normal cells, in contrast, any deeper-seated tumor cells wouldbe less affected by the topical polyamine composition, and would remainsensitive to the cancer therapeutic. Moreover, topical delivery of achemoprotective polyamine, which has a highly positive charge atphysiologic pH, should diminish any systemic exposure and limit theeffect on any tumor cells or normal host organ cells. Given the hosttoxicity that has been previously observed when polyamine analogs wereadministered systemically (Creaven, P. et al., Invest. New Drugs15:227-234, 1997; Streiff, Rand Bender, J., Invest. New Drugs 19:29-39,2001), this provides another important reason to avoid systemic deliveryof the chemoprotective polyamine molecules. The intended protection ofnormal tissue is achieved by an appropriate formulation ofchemoprotective polyamine in combination with an appropriate deliveryvehicle depending on the administration site (e.g. dermal/intradermal ormucosal). A pharmaceutical composition comprising a chemoprotectivepolyamine formulated with an appropriate delivery vehicle will haveutility in any normal cell type susceptible to the side effects ofcancer therapy that is accessible by topical delivery.

[0101] Thus, the chemoprotective polyamines of the invention areadministered topically (or locally) to protect patients from the sideeffects of cancer therapy. The term “topical” denotes the administrationof a drug intended to act locally rather than systemically. In thepresent invention, “topical” or “local” delivery is directed toepidermal and dermal cells of the skin and scalp (including cells lininghair follicles), as well as mucosal cells of the mouth, salivary glands,throat, gastrointestinal system and urogenital tract. For some of theselatter locations, compositions may be formulated for oral or nasaldelivery, or as suppositories. The goal of such delivery systems is tocontact these internal surfaces topically with the polyamine effectors.

[0102] The local delivery of drug molecules within the skin or mucousmembranes using a noninvasive delivery system has many attractions,including patient acceptability due to the noninvasiveness of theprocedure, avoidance of gastrointestinal digestion and disturbances, andfirst-pass metabolism of the delivered molecule. Topical delivery is notan efficient means for systemic drug delivery. It is estimated that onlybetween 1%-15% of a drug in most topical formulations is systemicallybioavailable. In preferred embodiments of the invention, less than 10%,preferably less than 5% and most preferably less than 1% of thepolyamine effector, provided topically e.g., dermal, intradermal,mucosal or GI epithelial delivery, move to reach the dermis and/or otherunderlying tissues.

[0103] Topical delivery vehicles can take the form of aqueous oraqueous:alcohol solutions, emulsions, creams, lotions, ointments, gelsor liposomes.

[0104] Solutions are the most traditional types of formulations fortopical dermal drugs, where the agent is solubilized in a solvent.Solvent-based systems are simple and effective constituents of topicaldelivery vehicles for some drugs. Alcohols are the most commonly usedsolvents for topical solutions. Typically, the drug is combined into awater and alcohol mixture. The alcohol content varies between 10-100%.Alcohols used include ethanol, propylene glycol, polyethylene glycols,methanol, or butanediol. Each of these types of alcohols is suitable foruse in the present invention; others not listed are also suitable, aswould be understood by one of skill in the art. High alcohol contentsolutions such as solutions of 70% ethanol in water or ones containing60% ethanol, 20% propylene glycol and 20% water, are particularly goodat penetrating the stratum corneum of the epidermis. Topical minoxidil,a hair regrowth treatment, uses the latter formulation as the deliveryvehicle.

[0105] Solution-based delivery systems are particularly suitable for thedelivery of small organic molecules. In a preferred embodiment of theinvention, particularly for administration of chemoprotective polyaminesto the epidermis, alcoholic solutions, as described above, are utilized.An aqueous alcohol-based delivery vehicle has been proven to be highlyeffective for topical administration of chemoprotective polyamines.Advantages of this delivery system include, ease of manufacturing, easeof application, fast drying, lack of residue on skin, and ease ofanalysis of active drug compound after formulation. Solution-typeformulations are typically administered using dropper bottles or asaerosols.

[0106] Emulsions form the basis of cream and lotion-type formulations.Typically, these formulations are colloidal dispersions composed of twoimmiscible phases; an oil phase and an aqueous phase with an emulsifier.Typical oils used in emulsions include stearyl alcohol, isopropyllanolate, isopropyl myristate, cetyl alcohol, and vitamin E. Emulsifiersare essentially surfactants that lower the surface tension of theimmiscible phases. Most emulsifiers tend to be fatty acid esters orstearates of glycerol, sorbitan, or polyoxyethylene (POE). Depending onthe location of the oil and water, emulsions are oil-in-water,water-in-oil or combinations thereof. The preparation of an emulsioncommonly requires some mechanical shear force with heat to mix theinternal and external phases. Most topical emulsions contain viscositybuilders such as natural gums (alginates, carrageenan, tragacanth,pectin, xanthan or collagen) at 1-5% to thicken the preparation. Higherpercentages of viscosity builders produce creams, a lower percentageform lotions. Complete formulations for emulsions (creams and lotions)generally include water, alcohol, propylene glycol, sodium laurylsulfate and white wax. In alternative formulations, they include water,alcohol, glycerol, phosphatidyl choline, lysophosphatidyl choline andtriglycerides. For administration of chemoprotective polyamines to theepidermis, emulsions are particularly well suited. Ease ofadministration, good local retention and slow release of drug are someof the attractive characteristics of emulsions for a topical deliverysystem.

[0107] Ointments are composed of fluid hydrocarbons meshed in a matrixof higher melting solid hydrocarbons. The hydrocarbon ointment base istypically petrolatum and white ointment. Ointments are prepared bymelting the base, followed by the addition of excipients, such asantioxidants to the fluid. The drug is then suspended into the ointmentby milling. Due to the high oil content, ointments tend to be greasy.Adding components, such as microcrystalline cellulose, which gives theointment a dry feel on the skin, can reduce greasiness. All ingredientslisted above for preparation of ointments are suitable for use in thepresent invention, as well as unlisted ingredients typically employedfor such purpose by one of skill in the art.

[0108] Gels are semisolids consisting of a gelling agent that ispenetrated with liquid solvent. The concentration and the molecularweight of the gelling agent affect the consistency of vehicleformulation. The gelling agent is a suspension of either large organicor small inorganic molecules. The large organic molecules consisting ofeither natural or synthetic polymers exist as randomly coiled chainsthat entangle and form the gel structure. Some common polymers of thiskind are natural gums, cellulose derivatives and acrylic acid polymers.Another class of these gels, called thermally sensitive gels, isprepared from poloxamers. In contrast, the small inorganic moleculesform the gel structure by forming a somewhat organized three-dimensionalnetwork. Common small inorganic polymers include colloidal solids foundin silica and clays. The nature of the solvent determines whether thegel is a hydrogel (water-based) or an organogel (non-aqueous solventbased). Gels are attractive topical delivery vehicles forchemoprotective polyamines because they are relatively easy to prepareand tend to have a long residence time at the site of applicationallowing the slow release of compound at the desired site. Allingredients listed above for preparation of gels are suitable for use inthe present invention, as well as unlisted ingredients typicallyemployed by one skilled in the art for such purpose.

[0109] Liposomes are vesicles consisting of amphipathic lipids arrangedin one or more concentric bilayers. When lipids are placed in aqueousmedium, the hydrophilic interaction of the lipid head groups with waterresults in the formation of multilamellar and unilamellar systems orvesicles which resemble biological membranes in the form of a sphericalshell. Liposomes may be small (0.025-0.05 um) to large multilamellarvesicles (0.05-10 um). Lipids used to prepare the liposomes includephospholipids, sphingolipids, glycosphingolipids, saturated glycerides,steroids (e.g., cholesterol) and synthetic phospholipids. Liposomes aretypically prepared by melting the lipid together in aqueous solvent withan emulsifier like POE. The drug is then added and the liposomes aregenerated through mixing or sonication. The drug is usually entrapped inthe vesicle structure. These basic liposomes are sometimes referred toas “conventional liposomes.” Several other types of liposomalpreparations exist, including (1) sterically stabilized liposomes, whichare surface coated with an inert hydrophilic polymer, such aspolyethylene glycol; (2) targeted liposomes, to which are attachedtargeting ligands, such as antibodies or fragments thereof, lectins,oligosaccharides or peptides (e.g., choleratoxin B (CTB) is used totarget liposomes to the gastrointestinal epithelium); and (3) reactiveor “polymorphic” liposomes, which change their phase and structure inresponse to a particular interaction (this group includes liposomessensitive to ions (pH, cations), heat and light, among other stimuli.

[0110] Liposomes are good vehicles for dermatological applications.Liposomal delivery offers certain advantages over more conventionalformulations, including: (1) reduced serious side effects andincompatability from undesirably high systemic absorption; (2)significantly enhanced accumulation of the delivered substance at thesite of administration due to high compatability of liposomes withstratum corneum; (3) ready incorporation of a wide variety ofhydrophilic and hydrophobic molecules into the skin; (4) protection ofthe entrapped compound from metabolic degradation; and (5) closeresemblance to the natural membrane structure and their associatedbiocompatibility and biodegradability. All ingredients listed above andfor preparation of various types of liposomes are suitable for use inthe present invention, as well as any other such ingredients typicallyemployed by one skilled in the art for such purpose.

[0111] In order to achieve efficient delivery of a chemoprotectivepolyamine into the skin, one embodiment of the invention includesvarious formulations of liposomes (phospholipid-based vesicles, cationicliposomes, nonionic liposomes, non ionic/cationic liposomes, pegylatedliposomes, PINC polymer, and propylene glycol and ethanol mixture(commonly used vehicle for administering minoxidil), and nonionicliposome/propylene glycol and ethanol mixtures. Reactive liposomes maybe preferred for other embodiments of the present invention. Inclusionof cationic amphiphiles as aminor component of liposomes facilitates theassociation with negatively charged solutes, the rapid binding ofliposomes to the cell surface, and the cellular uptake of liposomes.pH-sensitive liposomes have been developed to improve the efficiency ofthe cytoplasmic delivery of antitumor drugs, proteins, and nucleicacids. Most pH-sensitive liposomes have been prepared usingphosphatidylethanolamine (PE). PE alone does not form liposomes and isprone to form the inverted hexagonal phase (HII). However, liposomes canbe prepared by adding another bilayer-stabilizing, amphiphilic lipidcomponent to PE. Titratable amphiphiles having a carboxyl group havebeen used as a component for the preparation of pH-sensitive liposomes.Because the ability to stabilize a bilayer membrane by these titratableamphiphiles decreases under acidic conditions, destabilization resultsin the fusion of the liposomes. pH-sensitive liposomes are stable atphysiological pH, and are internalized by cells through an endocyticpathway, which exposes the liposomes to an acidic pH. Liposomes withinthe endosome are destabilized and possibly fuse with the endosomemembrane, resulting in release of their contents into the cytoplasmwithout degradation by lysosomal enzymes.

[0112] In other embodiments of the invention, sterically stabilized,inert liposomes are particularly suitable. In still other embodiments,targeted liposomes may be used to advantage.

[0113] For many applications, mucosal delivery will be used for deliveryof chemoprotective polyamines. Mucosal delivery defined here is thelocal delivery of polyamine effectors to the mucosa of the mouth, GI,and urogenital tract. Mucosally active drugs, can be formulated aseither solutions, emulsions or creams, ointments, gels or liposomesusing the ingredients described above. In addition, there are alsospecial excipients specifically designed for mucosal delivery. Thedescription, composition, and applicability of these major types ofmucosal delivery forms are set forth below. Each is considered suitablefor practice of various embodiments of the present invention.

[0114] In general, the structure of the mucosal surface is composed ofan outermost layer of stratified squamous epithelium, below which lie abasement membrane, a lamina propria followed by the submucosa as theinner-most layer. The mucosae of areas subject to mechanical stress suchas the gingivae or the hard palate are also keratinized, similar to theepidermis. Depending on the keratinization, the mucosa is somewhatpermeable. The permeability of oral mucosa is 4-4000 times greater thanthat of the skin. Permeability of intestinal mucosa is even greater. Thecells of the epithelia are surrounded by an intercellular groundsubstance, mucous, the principal components of which are complexes ofproteins, carbohydrates, lipids and ceramides. Primarily, specialmucous-secreting cells, called goblet cells, synthesize mucous. However,in the oral mucosa, most of the mucous is produced by the major andminor salivary glands. Mucous forms a strongly cohesive gel structurethat will bind to the epithelial cell surface as a gelatinous layer. Thepenetration of this mucous layer and the local retention of compoundbecause of its permeability must be achieved for effective mucosal drugdelivery. However, this route of administration is very important forthe delivery of compounds designed to protect mucosal surfaces fromcancer therapy. Since the mucosal surface is a common site in which manyof the unwanted side effects occur, the use of formulatedmucosally-active drugs designed to prevent these effects is warranted.

[0115] Issues to be considered with mucosal delivery are (1) low flux ordrug transport through the mucous layer and (2) poor retention andbioadhesion at the mucosal site. Mucosal permeation enhancers aredesigned to improve drug flux or penetration at the mucosal surface. Theuse of these enhancers can increase drug permeability by 100-fold ormore. Various permeation/absorption enhancers vary in molecular weightand physicochemical properties. In a preferred embodiment for mucosaldelivery, permeation enhancers are included in formulations for deliveryof chemoprotective polyamines to the mucosal surface. Most types ofenhancers are detergents that include: sodium glycocholate, sodiumtaurocholate, polysorbate 80, sodium lauryl sulfate, lauric acid, andvarious alkyl glycosides. Other examples of enhancers include: dextrins(cyclodextrin, dextran sulfate), fatty acids (phosphatidylcholine,lysophosphatidylcholine), heterocyclic compounds (azone), and smallmolecules (benzalkonium chloride, cetyltrimethylammonium bromide). Eachis contemplated for use in the present invention as are other unlistedingredients typically used for such purpose, as would be appreciated byone of skill in the art.

[0116] The addition of mucoadhesives to the formulation can improvelocal retention of mucosally delivered compounds. In another preferredembodiment for mucosal delivery, mucoadhesives are included in thepolyamine effector formulations of the invention. Mucoadhesive compoundsare primarily synthetic or natural polymers that can adhere to the wetmucosal surface. These include synthetic polymers such as monomericalpha cyanoacrylate, polyacrylic acid, hydroxypropyl methylcellulose,and poly methacrylate derivatives. Glue-like polymers include epoxyresins and polyurethanes. Naturally occurring mucoadhesives includechitosan, hyaluronic acid and xanthan gum. Each is contemplated for usein the present invention as are other unlisted ingredients typicallyused for such purpose, as would be appreciated by one of skill in theart.

[0117] Other delivery vehicles are also suitable for use in the presentinvention, particularly for administration of polyamine effectors to themucosa and lumen of the GI and urogenital tract. Nonlimiting examplesinclude: (1) oils such as vegetable oils or fish oils (which can beencapsulated into standard gel capsules); and (2) emulsions prepared,for example, by dispersing polyoxyethylene ethers, e.g., 10-stearylether (Brij 76) in aqueous buffer.

[0118] Other examples of delivery vehicles suitable for the GI orurogenital mucosa include biodegradable microparticles (preferably inthe range of 0.1-10 uM diameter) of polylactic polyglycolic acid, whichhave been used to deliver proteins to Caco-2 cells as an in vitro modelsystem for gastrointestinal uptake via oral drug delivery (Desai et al.,Pharm. Res. 14: 1568-1573, 1997). Significant uptake of proteins carriedby polystyrene particles into cells lining the small intestine of therat has been demonstrated (Hillery et al., J. Drug Targeting 2: 151-156,1994). Indeed, delivery of protein-containing microparticles has beenreported from the GI lumen all the way to the submucosal vasculature(Aphramaian et al., Biol. Cell 61: 69-76, 1987). Therefore, suchpolymeric microparticles are quite suitable for oral delivery ofpolyamine effectors to gastrointestinal epithelial cells, which arefound on the surface of the GI lumen.

[0119] Thus, chemoprotective polyamines are formulated as pharmaceuticalpreparations for topical or local administration to patients. Thefollowing sites of local administration of these pharmaceuticalpreparations are contemplated: oral, nasal, ophthalmic,gastrointestinal, urogenital and dermal (cutaneous). The term “patient”or “subject” as used herein refers to human or animal subjects (animalsbeing particularly useful as models for clinical efficacy of aparticular composition). Selection of a suitable pharmaceuticalpreparation depends upon the method of administration chosen, and may bemade according to protocols well known to medicinal chemists.

[0120] The pharmaceutical preparation comprising the compositions of theinvention are conveniently formulated for administration with abiologically acceptable medium such as water, buffered saline, alcohols,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol and the like), dimethyl sulfoxide (DMSO), oils, detergents,suspending agents or suitable mixtures thereof, as compatible with thespecific delivery vehicles described above. The concentration of aparticular composition in the chosen medium will depend on thehydrophobic or hydrophilic nature of the medium, in combination with thespecific properties of the delivery vehicle and active agents disposedtherein. As used herein, “biologically acceptable” or “pharmaceuticallyacceptable” refers to those compounds, materials, compositions, and/ordosage forms that are, within the scope of sound medical judgment,suitable for contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem complications commensurate with a reasonable benefit/risk ratio.

[0121] As used herein, “pharmaceutically acceptable salts” refer toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Thus, the term “acid addition salt” refers to the correspondingsalt derivative of a parent compound that has been prepared by theaddition of an acid. The pharmaceutically acceptable salts include theconventional salts or the quaternary ammonium salts of the parentcompound formed, for example, from inorganic or organic acids. Forexample, such conventional salts include, but are not limited to, thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Certain acidic orbasic compounds of the present invention may exist as zwitterions. Allforms of the compounds, including free acid, free base, and zwitterions,are contemplated to be within the scope of the present invention.

[0122] The topical formulation can contain a variety of excipients thatfunction to stabilize and solubilize the drug formulation, increasepermeation, and protect and aid in the application to the skin. Oil orwater-based excipients are primarily added to improve drug solubilityand spreadibility to the formulation. Surfactants may be added totopical formulations as detergents, solubilizers, emulsifiers, andwetting agents.

[0123] It will also be appreciated by persons of skill in the art thatpharmaceutical formulations of the invention may contain more than onechemoprotective polyamine. Various combinations of such agents may beuseful for certain applications, and formulations of such combinationswould be prepared according to the general guidelines set forth above.Moreover, one or more chemoprotective polyamines may be combined withother agents, such as other anti-proliferative agents or chemoprotectivedrugs, to provide a pharmaceutical formulation that is effective by twodifferent modes of action. An anti-proliferative agent suitable for suchuse is the cyclin-dependent kinase II inhibitor described in PCTapplication US00/05186, published Dec. 28, 2000 as WO 00/78289 orgenistein, an inhibitor of tyrosine protein kinase. A chemoprotectiveagent suitable for such use is resveratrol (trihydroxy-trans-stilbene).Several classes of “chemoprotective inducing agents” (agents that inducethe cell's endogenous defense processes) that may be combined with thechemoprotective polyamines of the invention are described in detail incommonly-owned, co-pending U.S. application Ser. No. 09/565,714, filedMay 5, 2000, and International Application No. PCT US01/14464, filed May4, 2001, the entireties of each of which are incorporated by referenceherein. Further, certain of those chemoprotective inducing agents alsopossess anti-proliferative activity.

[0124] The pharmaceutical preparation is formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit form,as used herein, refers to a physically discrete unit of thepharmaceutical preparation appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of the chemoprotectivepolyamine calculated to produce the desired protective effect inassociation with the selected pharmaceutical carrier. Procedures fordetermining the appropriate dosage unit are well known to those skilledin the art. As used herein, “therapeutically effective amount” refers toan amount of a compound as described herein that may be effective toinhibit, or treat the symptoms of particular disorder or side effect.The term “prophylactically effective amount” refers to an amount of acompound as described herein that may be effective to prevent, inhibit,or diminish the onset the symptoms of a particular disorder or sideeffect.

[0125] Dosage units may be proportionately increased or decreased basedon the height and weight of the patient. Appropriate concentrations forachieving protection of a target cell population or tissue from thetoxic effect of a particular chemotherapeutic agent may be determined bydosage concentration curve calculations, as known in the art.

[0126] As one example, for topical applications, the chemoprotectivepolyamine may be used at concentrations ranging from 1-100 mM in anappropriate carrier (e.g., alcohol solvent) applied to the scalp orother dermal site. This dosage is arrived at from results of experimentsusing a rodent model and the range of dosages is a function of resultsobtained from experiments using several different molecules that rangedin dose effectiveness. The volume of material applied to the skin rangesby size of surface area to be covered; e.g., scalp treatment for youngchildren requiring 3-5 ml, the amount being increased in adults to 10-20ml per application.

[0127] As another example, for gastrointestinal administration, the oraldose of the chemoprotective polyamine in an appropriate medium (e.g.,solvent, liposome emulsion) is normalized to the lumenal surface area ofthe stomach and duodenum. This would assume that the patient consumesthe material on an empty stomach upon rising in the morning.

[0128] The pharmaceutical preparation comprising the compositions of theinvention may be administered at appropriate intervals, before, during,or after a regimen of chemotherapy and/or radiotherapy. The appropriateinterval in a particular case would normally depend on the nature of thechemotherapy or radiotherapy and the cell population targeted forprotection.

[0129] For instance, for prevention of chemotherapy-induced alopecia,solvents, liposomes or other delivery vehicles containing thechemoprotective polyamine can be further formulated to be delivered,(e.g., as a topical cream, or gel) to the scalp of a patient prior toscheduled administration of chemotherapy. By protecting the epithelialcells that line the exposed surface of hair follicles from thechemotherapy drug, the loss of hair commonly associated with cancerchemotherapy is prevented. Likewise, for the treatment ofradiation-induced dermatitis, the chemoprotective polyamine can befurther formulated as a gel, ointment or cream containing moisturizers.This would further protect the epidermis from radiation damage. Thetopical formulation preferably is initiated several days prior to thecancer therapy, to ensure that the epithelial and mucosal cells areadequately treated. The formulation may then continue to be appliedduring the course of chemotherapy.

[0130] For protection of the gastrointestinal epithelium, thechemoprotective polyamine is formulated to be delivered by mouth to apatient prior to scheduled administration of cancer therapy.Administration of the protective formulation in the 1-5 days prior toradiotherapy or the infusion of the chemotherapeutic agent thus confersprotection to susceptible mucosal epithelial cells. For example, thepatient would be instructed to consume a “shake” containing thechemoprotective polyamine in an orally acceptable solution or liposomeemulsion before breakfast in the morning, in the 1-5 days precedingchemotherapy. This would allow the chemoprotective polyamine to bepresent when the chemotherapy drugs or radiotherapy act on the GImucosal epithelium.

[0131] The examples that follow are included to aid in a more completeunderstanding of the present invention. The examples do not limit theinvention disclosed and claimed herein in any fashion. Referencenumerals are to the reaction schemes described above. All purificationcolumns were carried out using silica gel (230-400 mesh) with eluantnoted. Silica gel plates (250 micron) were used for all thin layerchromatography (TLC) with the appropriate solvent system noted.

EXAMPLE 1 Preparation of Compounds Used in Synthetic Schemes

[0132] Scheme 1:

[0133] Compound 2:2 M ethylamine (compound 1) in tetrahydrofuran wasstirred in a pressure bottle at <0° C. and mesitylene sulfonyl chloride(3 molar equivalents wrt ethylamine) was added in portions so that thetemperature did not exceed 10° C. Dichloromethane and triethylamine wereadded and the pressure bottle sealed. The reaction was stirred in a 30°C. water bath for one hour and at RT for 30 minutes. The reactionprogress was monitored by TLC using 8:2 heptanes: ethyl acetate as themobile phase. Water was added and the organic layer was separated, thewater layer was extracted once with dichloromethane, the combinedorganic layers were washed twice with water and condensed under vacuum.The product was used without further purification.

[0134] Compound 3: NaH (1.2 molar equivalents wrt compound A) wasstirred, under N₂, at 10° C. and dimethylformamide was added. Compound 2dissolved in tetrahydrofuran was added and stirred until the evolutionof H₂ gas ceased. Bromobutyl (or any N-alkyl depending on desireddistance between amines)-phthalimide (1.1 molar equivalents wrt tocompound 2) was added in one portion and NaI was added. The reaction washeated to 60° C. and the progress monitored after several hours by TLCusing 7:3 heptanes: ethyl acetate as the mobile phase. The reactioncontents were condensed under vacuum and dissolved in ethyl acetate andwater. The organic layer was separated, the aqueous layer was extractedwith ethyl acetate, and the combined organic layers were washed withdilute brine and condensed under vacuum. The product was used withoutfurther purification.

[0135] Compound 4: Ethanol was heated to 70° C. and compound B dissolvedin hot ethanol was added. Hydrazine hydrate (2.5 molar equivalents wrtcompound 3) was added all at once and the reaction was stirred at 70° C.overnight. The reaction progress was monitored by TLC using 6:4heptanes: ethyl acetate as the mobile phase. The completed reaction wascooled on ice and a white precipitate formed. The precipitate wasremoved by filtration and the filtrate condensed under vacuum. Theresulting semisolid was dissolved in dichloromethane and water. Theorganic layer was separated, the aqueous layer was extracted withdichloromethane and the combined organic layers were washed with waterand condensed under vacuum. The product was purified by columnchromatography using silica gel and 90:9:1 dichloromethane: methanol:ammonium hydroxide as the eluant.

[0136] Compound 5: Mesitylene sulfonyl chloride (1.1 molar equivalentswrt compound 4) dissolved in dichloromethane was stirred, under N₂, at10° C. Compound C dissolved in dichloromethane was slowly added so thatthe temperature did not exceed 15° C. The reaction was cooled to 10° C.and triethylamine (1.2 molar equivalents wrt compound 4) was added. Thereaction was stirred at RT for several hours. The progress was monitoredby TLC using 1:1 heptanes: ethyl acetate as the mobile phase. Thereaction was quenched by adding water and stirring for 20 minutes. Theorganic layer was separated, the aqueous layer was extracted with ethylacetate then dichloromethane, and the combined organic layers werewashed with water and condensed under vacuum. The product was purifiedby column chromatography using silica gel and 6:4 heptanes: ethylacetate as the eluant.

[0137] The polyamine side chains are elongated by repeating steps 2-4until the desired length is reached.

[0138] Scheme 2:

[0139] Compound 16: Dihydroxyacetone dimer, compound 15, was stirred indimethylformamide, under N₂, at 2° C. Imidazole (5.02 molar equivalentswrt. Compound 15) then tert-butyl dimethylsilyl chloride (4.99 molarequivalents wrt compound 15) were added. The reaction was stirred at RTfor 2 hours. Ice water was added and the reaction stirred for 20minutes. The organic layer was separated, the aqueous layer extractedtwo times with ethyl acetate, the combined organic fractions were washedwith dilute brine, dried over anhydrous MgSO4, filtered, and condensedunder vacuum to yield brown oil. The oil was purified by columnchromatography using silica gel and 97:3 heptanes: ethyl acetate then95:5 heptanes: ethyl acetate as the eluant.

[0140] Compound 17: NaH (1.1 molar equivalents wrt compound 1) wasstirred, under N₂, in an ice bath and toluene was added. Triethylphosphonoacetate (1.01 molar equivalents wrt compound 16) was slowlyadded so that the temperature did not exceed 10° C. The reaction wasstirred on ice until all observed effervescence stopped. The reactionwas removed from the ice bath and compound 16 (bis-OTBS acetone) wasadded drop-wise. The reaction was stirred at RT for 1.5 hours andethanol was added to dissolve a precipitate that had formed. Water wasadded to quench the reaction. The organic layer was separated, theaqueous layer extracted once with ethyl acetate, and the combinedorganic layers were washed with brine and dried over anhydrous MgSO₄.The organic solution was filtered and condensed under vacuum to yieldyellow oil. The oil was purified by column chromatography using silicagel and 98:2 heptanes: ethyl acetate.

[0141] Compound 18: Compound 2 was stirred in ether and cooled, underN₂, to −80° C. in an acetone/dry ice bath. Diisobutyl aluminum hydride(1.5 molar equivalents wrt compound 17) was added drop wise. Thereaction was removed from the acetone/dry ice bath, warmed to RT, andstirred at RT for 50 minutes. The reaction was cooled in an acetone/dryice bath and water was added drop wise to quench the reaction. Theacetone/dry ice bath was removed and 20% NaOH (molar equivalents wrtcompound 17), dichloromethane, and Rochelle salt (KNa tartratetetrahydrate) were added. The organic layer was separated, the aqueouslayer extracted two times with dichloromethane, and the organicfractions were combined, washed with water and dried first with K₂CO₃and then MgSO₄. The dried organics were filtered and condensed undervacuum to yield clear oil. The clear oil was purified by columnchromatography using silica gel and 9:1 heptanes: ethyl acetate as theinitial eluant then changing to 8:2 heptanes: ethyl acetate.

[0142] Compound 19: Compound 18 was stirred in dichloromethane, underN₂, and cooled to below 0° C. in an acetone/ice bath. Triethylamine (1.2molar equivalents wrt compound 18) was added and the reaction cooled tobelow 0° C. Methane sulfonyl chloride (1.3 molar equivalents wrtcompound 18) was added slowly while monitoring the temperature to assurethat it did not exceed 5° C. The reaction stirred cold for 1 hour thendichloromethane and water were added. The organic layer was separated,the aqueous layer extracted with dichloromethane, the combined organiclayers were dried with K₂CO₃ and MgSO₄, filtered and condensed undervacuum to yield the mesylate intermediate. The product was used withoutfurther purification.

[0143] Compound 20: NaH (1.25 molar equivalents wrt compound 18) wasstirred with dimethyl formamide, under N₂, and a polyamine side chain(1.15 molar equivalents wrt compound 18), of chosen length, dissolved intetrahydrofuran was slowly added. The reaction stirred at RT until theevolution of H₂ gas ceased. Starting material mesylate was slowly added(compound 4, step 1 product) and stirred at RT for several hours. Uponcompletion, as evidenced by TLC, the reaction contents were condensedunder vacuum. The crude semi-solid was dissolved in ethyl acetate andwater. The organic layer was separated; the aqueous layer extractedtwice with ethyl acetate, the combined organic layers were washed withwater and condensed under vacuum. The product was purified by columnchromatography using silica gel and 75:25 heptanes: ethyl acetate as theeluant.

[0144] Compound 21: Compound 20 was stirred in methanol at RT.Concentrated HCl (2 molar equivalents wrt compound 20) was slowly added.The reaction stirred at RT for 30 minutes or until reaction was completeas evidenced by TLC with 60:40 heptanes: ethyl acetate as the mobilephase. The reaction contents were condensed under vacuum and purified bycolumn chromatography using silica gel and 95:5 dichloromethane:methanol as the eluant.

[0145] Compound 22: Compound 21 diol was stirred in dichloromethane,under N₂, in an ice/MeOH bath. Benzoyl Chloride (1.03 molar equivalentswrt compound 21) was added. Once the reaction reached <10° C., pyridine(1.04 molar equivalents wrt compound 21) was slowly added. The reactionwas stirred in the ice/methanol bath for 1 hour and completeness wasdetermined by TLC using 1:1 heptanes: ethyl acetate as the mobile phase.Once reaction was complete, water was added and the reaction stirred for15 minutes in the ice/methanol bath. The organic layer was separated;the aqueous layer extracted with dichloromethane, the combined organiclayers were washed once with water, dried over anhydrous MgSO₄, filteredand condensed under vacuum. The product was purified by columnchromatography using silica gel and 7:3 heptanes: ethyl acetate as theeluant.

[0146] Compound 23: Compound 22 was stirred in toluene, under N₂, at <5°C. Phosphorus tribromide (1.1 molar equivalents wrt compound 22) wasslowly added. The reaction was removed from the ice bath and stirred atRT for 30 minutes or until the reaction was complete as determined byTLC using 95:5 dichloromethane: methanol as the mobile phase. Uponcompletion the reaction was returned to the ice bath, water was slowlyadded, and the reaction was stirred for 15 minutes. The organic layerwas separated, the aqueous layer extracted two times with ethyl acetate,the combined organic layers were washed with 2% (w:v) NaHCO3 and thenbrine, dried over K₂CO₃ and MgSO₄, filtered and condensed under vacuum.The product was used without further purification.

[0147] Compound 24: NaH (1.2 molar equivalents wrt compound 23) wasstirred in dimethyl formamide, under N₂, at RT and a polyamine sidechain (1.2 molar equivalents wrt compound 23), of chosen length,dissolved in tetrahydrofuran was added slowly. The reaction stirred atRT until the evolution of H₂ gas ceased. Compound 23, dissolved intetrahydrofuran, was slowly added and the reaction was stirred at RT forseveral hours. Reaction completeness was determined by TLC using 80:20toluene: ethyl acetate as the mobile phase. The reaction was condensedunder vacuum; the crude was dissolved in ethyl acetate and water. Theorganic layer was separated, the aqueous layer was extracted with ethylacetate, and the combined organic layers were washed with brine, andcondensed under vacuum. The product was used without furtherpurification.

[0148] Compound 25: Compound 24 was stirred in tetrahydrofuran, underN₂, at RT. Methanol then sodium methoxide (1.5 molar equivalents wrtcompound 24) were added and the reaction was stirred at RT for 30minutes. Reaction completeness was determined by TLC using 80:20toluene: ethyl acetate as the mobile phase. Concentrated HCl (molarequivalents wrt sodium methoxide) was added to neutralize the sodiummethoxide and the reaction contents were condensed under vacuum. Ethylacetate and water were added to the crude product. The organic layer wasseparated, the aqueous layer washed once with ethyl acetate and oncewith dichloromethane, the combined organic layers dried with NaSO₄,filtered and condensed under vacuum. The product was purified by columnchromatography using silica gel and 8:2 toluene: ethyl acetate as theeluant.

[0149] Scheme 3:

[0150] Compound 28: Compound 26 was stirred in dichloromethane, underN₂, at −10° C. in an ice/methanol bath. Triethylamine (2 molarequivalents wrt to compound 26) was added and the reaction was againcooled to −10° C. Methane sulfonyl chloride (2.5 molar equivalents wrtcompound 26) dissolved in methylene chloride was added slowly and thereaction stirred cold for 1 hour. Reaction completeness is monitored byTLC using 8:2 heptanes: ethyl acetate. Water was slowly added to quenchthe reaction. The organic layer was separated, the water layer extractedwith dichloromethane, the combined organic layers were washed with brineand condensed under vacuum. The reactive intermediate was usedimmediately without further purification.

[0151] Compound 29: Potassium thioacetate (2.5 molar equivalents wrtcompound 26) in dimethylformamide was stirred, under N₂, at RT. Compound28 mesylate in dimethylformamide was slowly added and the reaction wasstirred overnight. The reaction was condensed under vacuum and thesolids dissolved in ethyl acetate and water. The organic layer wasseparated, the aqueous layer back extracted with ethyl acetate, thecombined organic layers were washed with brine and condensed undervacuum. The product was purified by column chromatography using silicagel and 8:2 toluene: ethyl acetate as the eluant.

[0152] Compound 31: NaH (1.25 molar equivalents wrt compound 26) wasstirred, under N₂, at RT and dimethylformamide was added. Mesitylenemethyl sulfonamide dissolved in tetrahydrofuran was slowly added and thereaction was stirred until the evolution of H₂ gas ceased. Compound 28mesylate dissolved in tetrahydrofuran was slowly added and the reactionwas stirred overnight. The reaction was condensed under vacuum and thesolids dissolved in ethyl acetate and water. The organic layer wasseparated, the aqueous layer extracted with ethyl acetate, the combinedorganic layers were washed with brine and condensed under vacuum. Theproduct was purified by column chromatography using silica gel and 8:2toluene: ethyl acetate as the eluant.

[0153] Compound 33: NaH (1.25 molar equivalents wrt compound 26) wasstirred, under N₂, at RT and dimethylformamide was added. Mesitylenedimethyl sulfonamide dissolved in tetrahydrofuran was slowly added andthe reaction stirred until the evolution of H₂ gas ceased. Compound 28mesylate dissolved in tetrahydrofuran was slowly added and the reactionwas stirred overnight. The reaction was condensed under vacuum and thesolids dissolved in ethyl acetate and water. The organic layer wasseparated, the aqueous layer extracted with ethyl acetate, the combinedorganic layers were washed with brine and condensed under vacuum. Theproduct was purified by column chromatography using silica gel and 8:2toluene: ethyl acetate as the eluant.

[0154] Compound 35: NaH (1.25 molar equivalents wrt compound 26) wasstirred, under N₂, at RT and dimethylformamide was added. Mesityleneethyl sulfonamide dissolved in tetrahydrofuran was slowly added and thereaction stirred until the evolution of H2 gas ceased. Compound 28mesylate dissolved in tetrahydrofuran was slowly added and the reactionstirred overnight. The reaction was condensed under vacuum and thesolids dissolved in ethyl acetate and water. The organic layer wasseparated, the aqueous layer extracted with ethyl acetate, the combinedorganic layers were washed with brine and condensed under vacuum. Theproduct was purified by column chromatography using silica gel and 8:2toluene: ethyl acetate as the eluant.

[0155] Removal of Mesitylene Protective Groups:

[0156] Compounds 30, 32, 34, and 36: Starting material was stirred indichloromethane at RT and phenol (11 molar equivalents per mesitylenegroup) was added. 30% HBr in acetic acid was slowly added (13 molarequivalents per mesitylene group) and the reaction was tightly sealedand stirred for 24-72 hours at RT. Water was added and the reactionstirred for 30 minutes at RT. The organic layer was separated, theaqueous layer was washed five times with dichloromethane, and the waterlayer was condensed under vacuum. 30% NaOH was added to the oil andstirred for several minutes to make the free base. Dichloromethane wasadded and stirred for several more minutes. The organic layer wasseparated, the water layer was extracted five times withdichloromethane, and the combined organic layers were condensed undervacuum. The HCl salt was made by stirring the free base in ethanol andslowly adding concentrated HCl (4 molar equivalents per free amine). Thereaction was condensed under vacuum and the solids were recrystallizedin a hot ethanol/water mixture.

EXAMPLE 2 Biological Assay for Efficacy in Preventing Alopecia

[0157] The efficacy of chemotherapeutic polyamines in reducing orpreventing chemotherapy-induced alopecia in a rat model was examined.This animal model mimics many of the features found inchemotherapy-induced alopecia seen in humans and is considered aclinically relevant model for testing novel therapeutics.

[0158] Induction of alopecia by cytoxan (CTX). Lactating Sprague Dawleymother rats with rat pups were purchased from Harlan Sprague Dawley(Indianapolis, Ind.). The mother rats were given food and water adlibitum. The rats pups were tested in the model of chemotherapy-inducedalopecia described by Hussein A. M. et al., Science: 249, 1564 (1990).Cytoxan (CTX), a chemotherapeutic widely used in the treatment ofcancer, was used to induce alopecia in the rats. A common side effect ofcytoxan in patients is alopecia. Cytoxan was purchased from SigmaChemicals Co. (St. Louis, Mo.). To produce CTX-induced alopecia, 7 to 10day old rat pups were injected i.p. with 35 mg/kg of CTX prepared inphosphate-buffered saline. It was observed that 35 ug/gm of CTX wassufficient to induce 100% hair loss approximately 7 days after cytoxanchallenge.

[0159] Chemoprotective polyamines of the invention were prepared in adelivery vehicle consisting of from 60-100% ethanol in water, dependingon the solubility of the compound. The compounds in ethanol/watersolution from 50-150 μl in volume were topically administered to thebacks of the pups once per day before and after CTX challenge. Using amicropipette, the formulation was applied to approximately ² cm2 sectionof skin to the backs of the rat pups. Specifically, the pups weretreated once daily for the 4-5 days before CTX challenge, once on theday of CTX challenge and once daily for 5 days afterwards. Controlgroups consisted of pups receiving only delivery vehicle. Control groupstreated with delivery vehicle were tested as part of every treatmentstudy. Two or more animals were tested per group in both the control andtest groups.

[0160] Approximately 7 to 10 days after CTX treatment, the pups wereevaluated for alopecia. Hair loss was evaluated using a modifiedalopecia-scoring index described by Chen G. et al., Int. J. Cancer: 75,303 (1998). A score of 0=no hair loss; a score of 1=10-30% hair loss; ascore of 2=40-60% hair loss; a score of 3=70-90% hair loss; and a scoreof 4=100% hair loss.

EXAMPLE 3 Biological Assay for Efficacy in Preventing Dermatitis

[0161] To determine efficacy of chemoprotective polyamines in preventingradiation-induced dermatitis, adult rats were topically treated with thecompounds before and after radiation treatment. Rats were exposed tomedically relevant levels of radiation that could induce clinicalradiation dermatitis. Sprague Dawley rats (Harlen Spraque Dawley) at 4-6weeks-old were anesthetized with sodium pentobarbital at 40 mg/kg bodyweight (Sigma, St. Louis, Mo.) prior to radiation exposure. A defined,depilated area on the backs of rats was irradiated using a Mark I, Model30, Cs 137 irradiator (J. L. Sheppard & Associates). Briefly, the backwas stripped of hair to expose the skin using a 1:1 rosin/beeswaxmixture. The rest of the body was protected from radiation exposureusing a lead shield. A dose response study was initially preformed toreproduce relevant dermatitis that matched the Grade (I-IV) scale usedto score the severity of radiation-induced dermatitis in the clinicalsetting. Radiation doses of 5-7 Gray (1 Gray (Gy)=100 mrem) producedGrade I dermatitis within 8-10 days. Radiation doses of 7-10 Gy producedGrade II dermatitis within 8-10 days. After 8-10 days, severe radiationdermatitis was produced at 20-25 Gy (Grade III dermatitis) or at 30-35Gy (Grade IV). Radiation dermatitis of Grade II-III was considered mostclinically relevant, so a radiation challenge dose of 15 Gy in the ratswas used. The stripped back region on the rats was treated topicallywith chemoprotective polyamine once daily for 5 days before and 5 daysafter radiation challenge.

[0162] The polyamines were prepared in a delivery vehicle, consisting offrom 60-100% ethanol in water, depending on the solubility of thecompound. The compounds in ethanol/water solution from 100-150 μl involume were topically administered to the stripped region. Rats treatedwith only the delivery vehicle served as controls. Eight to ten dayspost-radiation challenge, the rats were evaluated for dermatitis using amodified scoring scale described by Masuda K. et al. Int. J. RadiationOncol. Biol. Phys: 12, 1645 (1986). Dermatitis score of 0=normal,1=slight redness, scaly skin with no focal lesions, 2=moderate redness,breakdown of larger area, some small focal lesions, 3=skin very red,breakdown of most of the irradiated area, large ulcers and crustylesions, 4=skin very red, breakdown of the entire irradiated area,severe exudation and large crusty lesions.

EXAMPLE 4 Radiation-induced Mucositis Model in Hamsters

[0163] The model for radiation-induced oral mucositis was developed forthe purpose of screening and identifying effective polyamines useful fortreatment. The model used in this example was derived from the oralmucositis model described by Sonis S. T. et al. (Oral Oncology36:373-381, 2000). Male golden Syrian hamsters (70-95 gram, 35-42 days,Charles River Laboratories, Wilmington, Mass.) were used. Animals wereindividually numbered, housed in small groups and fed and watered adlibitum. Hamsters were anesthetized with sodium pentobarbital (80 mg/kgbody weight, Sigma, St. Louis, Mo.). The left buccal cheek pouch waseverted and secured. A protective lead shield covered the remainder ofthe animal. Subsequently, the cheek pouch was irradiated with a singledose of radiation from 10 to 50 Gy delivered to the targeted mucosa inthe 137 Cs Irradiator. Starting 10 to 12 days after radiation, theseverity of mucositis was assessed every two days. The severity level ofmucositis was evaluated using a modified mucositis scoring systemdescribed by Sonis S. T. et al. (Oral Oncology 36:373-381, 2000) Thescoring system was as follows:

[0164] 0=Pouch completely healthy. No erythema or vasodilatation.

[0165] 1=Erythema.

[0166] 2=Severe erythema, vasodilatation

[0167] 3=Severe erythema and vasodilatation. Superficial erosion onradiated pouch surface area.

[0168] 4=Formation of ulcers in one or more places. Cumulative ulcerformation about up to 50% of radiated pouch surface area. Diminishedpliability of mucosa

[0169] 5=Virtually more then 50% or complete ulceration of the radiatedpouch mucosa. Loss of pliability.

[0170] Manifestations of radiation-induced mucositis were observed byday 12. The hamster buccal pouches were evaluated for the presence ofmucositis and photographed every two days from day 12 to day 20.Mucositis was found to increase in severity, reaching a peak at day 16.An obvious dose response of radiation was seen, and the grades ofmucositis at day 16 were scored as: Treatment Mucositis Grade*  0 Gy 010 Gy 1 20 Gy 2 30 Gy 2.5 40 Gy 4 50 Gy 5 # more than 50% or completeulceration of the radiated pouch mucosa; loss of pliability.

[0171] The present invention is not limited to the embodiments describedand exemplified above, but is capable of variation and modificationwithin the scope of the appended claims.

What is claimed:
 1. A compound of Formula I:

wherein: each Z is independently A or R¹; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R⁵; each R¹ is independently C₃₋₈ alkylene;each R², R³, R⁶, and R⁷ is independently C₁₋₆ alkylene; R⁴ is H or loweralkyl; R⁵ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷-SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.
 2. A compound of claim 1, wherein each A is independently:


3. A compound of claim 1, wherein each A is independently:


4. A compound of claim 2, wherein Y is H or R³-D.
 5. A compound of claim4, wherein Y is H.
 6. A compound of claim 4, wherein Y is R³-D.
 7. Acompound of claim 5, wherein X is D.
 8. A compound of claim 5, wherein Xis R²-D.
 9. A compound of claim 6, wherein X is D.
 10. A compound ofclaim 6, wherein X is R²-D.
 11. A compound of claim 2, wherein k is aninteger from 2 to
 8. 12. A compound of claim 2, wherein k is an integerfrom 9 to about
 16. 13. A compound of claim 2, wherein k is an integerfrom 9 to
 12. 14. A compound of claim 2, wherein k is
 2. 15. A compoundof claim 2, wherein k is
 3. 16. A compound of claim 2, wherein k is 4.17. A compound of claim 2, wherein k is
 5. 18. A compound of claim 2,wherein k is
 6. 19. A compound of claim 2, wherein k is
 7. 20. Acompound of claim 2, wherein k is
 8. 21. A compound of claim 2, wherein:k is the integer 2; each R¹ is butylene; X is D, D is-NR⁴R⁵, R⁴ is H,and R⁵ is ethyl; and Q is ethyl.
 22. A compound of claim 2, wherein: kis the integer 8; each R¹ is butylene; X is D, D is-NR⁴R⁵, R⁴ is H, andR⁵ is ethyl; and Q is ethyl.
 23. A compound of claim 2, wherein: k isthe integer 2; each R¹ is butylene; X is D, and D is —SH; and Q isethyl.
 24. A compound of claim 2, wherein: k is the integer 4; each R¹is butylene; X is D, and D is —SH; and Q is ethyl.
 25. A compound ofclaim 2, wherein: k is the integer 6; each R¹ is butylene; X is D, and Dis —SH; and Q is ethyl.
 26. A compound of claim 2, wherein: k is theinteger 8; each R¹ is butylene; X is D, and D is —SH; and Q is ethyl.27. A compound of claim 2, wherein: k is the integer 4; each R¹ isbutylene; X is D, D is —NR⁴R⁵, R⁴ is H, and R⁵ is methyl; and Q isethyl.
 28. A compound of claim 4 wherein Q is H or lower alkyl.
 29. Acompound of claim 3, wherein Y is H or R³-D.
 30. A compound of claim 29,wherein Y is H.
 31. A compound of claim 29, wherein Y is R³-D.
 32. Acompound of claim 30, wherein X is D.
 33. A compound of claim 30,wherein X is R²-D.
 34. A compound of claim 31, wherein X is D.
 35. Acompound of claim 31, wherein X is R²-D.
 36. A compound of claim 3,wherein k is an integer from 2 to
 8. 37. A compound of claim 3, whereink is an integer from 9 to about
 16. 38. A compound of claim 3, wherein kis an integer from 9 to
 12. 39. A compound of claim 3, wherein k is 2.40. A compound of claim 3, wherein k is
 3. 41. A compound of claim 3,wherein k is
 4. 42. A compound of claim 3, wherein k is
 5. 43. Acompound of claim 3, wherein k is
 6. 44. A compound of claim 3, whereink is
 7. 45. A compound of claim 3, wherein k is
 8. 46. A compound ofclaim 29 wherein Q is H or lower alkyl.
 47. A compound of claim 3wherein J is a single bond.
 48. A compound of claim 3 wherein J is—CH(Y)—.
 49. A pharmaceutical preparation for reducing or preventinghair loss, dermatitis, mucositis or gastrointestinal distress caused bytreatment with a chemotherapeutic agent or radiation therapy, whichcomprises at least one compound of Formula I and a topical deliveryvehicle for locally delivering the compound to dermal or mucosal cellsof skin, scalp, mouth, nasoesophageal, gastrointestinal or urogenitalsystem, wherein Formula I is

wherein: each Z is independently A or R¹; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R⁵; each R¹ is independently C₃₋₈ alkylene;each R², R³, R⁶, and R⁷ is independently C₁₋₆ alkylene; R⁴ is H or loweralkyl; R⁵ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷—SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.
 50. The pharmaceutical preparation of claim 49, which furthercomprises at least one other agent that reduces or prevents hair loss,dermatitis, mucositis or gastrointestinal distress caused by treatmentwith a chemotherapeutic agent or radiation therapy.
 51. Thepharmaceutical preparation of claim 50, wherein the other agent is ananti-proliferative agent.
 52. The pharmaceutical preparation of claim50, wherein the other agent is a chemoprotective inducing agent.
 53. Thepharmaceutical preparation of claim 50, wherein the other agent is afree radical scavenger.
 54. The pharmaceutical preparation of claim 49,wherein the topical delivery vehicle comprises one or more of liposomes,lipid droplet emulsions, oils, aqueous emulsions of polyoxyethyleneethers, aqueous alcohol mixtures, aqueous ethanol mixtures containingpropylene glycol, aqueous ethanol mixtures containing phosphatidylcholine, lysophosphatidyl choline and triglycerides, xanthan gum inaqueous buffer, hydroxypropymethylcellulose in aqueous buffer or aqueousalcohol mixtures, diethylene glycol monoethyl ether in aqueous buffer,and biodegradable microparticles.
 55. The pharmaceutical preparation ofclaim 54, formulated for topical delivery to skin or hair follicles,wherein the delivery vehicle comprises an aqueous alcohol mixture. 56.The pharmaceutical preparation of claim 55, wherein the delivery vehiclefurther comprises propylene glycol.
 57. The pharmaceutical preparationof claim 56, formulated as a cream, lotion, ointment or gel.
 58. Thepharmaceutical preparation of claim 54, formulated for topical deliveryto the oral cavity or naso-esophageal passages, wherein the deliveryvehicle comprises a mucoadhesive substance.
 59. The pharmaceuticalpreparation of claim 58, formulated as an aerosol, oral rinse, ointmentor gel.
 60. The pharmaceutical preparation of claim 54, formulated forvaginal or rectal delivery, wherein the delivery vehicle comprises amucoadhesive substance.
 61. The pharmaceutical preparation of claim 60,formulated as a cream, ointment, lotion, gel, foam or suppository. 62.The pharmaceutical preparation of claim 54, formulated for topicaldelivery to the gastrointestinal tract, wherein the delivery vehiclecomprises one or more of nonionic liposomes and mucoadhesive substances.63. The pharmaceutical preparation of claim 62, formulated as a liquidfor coating the surface of the gastrointestinal tract.
 64. A method forreducing or preventing hair loss dermatitis, mucositis orgastrointestinal distress in a patient undergoing treatment with achemotherapeutic agent or radiation therapy, which comprisesadministering to the patient a prophylactically or therapeuticallyeffective amount of a pharmaceutical preparation comprising at least onecompound of Formula I and a topical delivery vehicle for locallydelivering the compound to dermal or mucosal cells of skin, scalp,mouth, nasoesophageal, gastrointestinal or urogenital system, whereinFormula I is:

wherein: each Z is independently A or R¹; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R⁵; each R¹ is independently C₃₋₈ alkylene;each R², R³, R¹, and R⁷ is independently C₁₋₆ alkylene; R⁴ is H or loweralkyl; R⁵ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷—SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.
 65. The method of claim 64, wherein the pharmaceuticalpreparation is administered beginning at least one day prior tochemotherapy or radiation therapy.
 66. The method of claim 65, whereinthe pharmaceutical preparation is administered beginning at least fivedays prior to chemotherapy or radiation therapy.
 67. The method of claim64, wherein the pharmaceutical preparation is administered afterinitiation of chemotherapy or radiation therapy.
 68. The method of claim64, wherein the pharmaceutical preparation is administered throughout acourse of chemotherapy or radiation therapy.
 69. The method of claim 64,wherein the pharmaceutical preparation is administered followingtermination of a course of chemotherapy or radiation therapy.
 70. Themethod of claim 64, which further comprises administering to the patientat least one other agent that reduces or prevents hair loss, dermatitis,mucositis or gastrointestinal distress caused by treatment with achemotherapeutic agent or radiation therapy.
 71. The pharmaceuticalpreparation of claim 70, wherein the other agent is ananti-proliferative agent.
 72. The pharmaceutical preparation of claim70, wherein the other agent is a chemoprotective inducing agent.
 73. Thepharmaceutical preparation of claim 70, wherein the other agent is afree radical scavenger.
 74. A method of treating cancer that increases apatient's tolerance to high doses of a chemotherapeutic'agent orradiation therapy, the method comprising: a) administering the high doseof the chemotherapeutic agent or radiation therapy to the patient; andb) administering one or more pharmaceutical preparations for reducing orpreventing one or more of chemotherapy- or radiation therapy-inducedhair loss, dermatitis, mucositis or gastrointestinal distress, in anamount and for a time to reduce or prevent the one or more of thechemotherapy- or radiation therapy-induced hair loss, dermatitis,mucositis or gastrointestinal distress, thereby increasing the patient'stolerance to the high dose of the chemotherapeutic agent or radiationtherapy, wherein the pharmaceutical preparation comprises compound ofFormula I and a topical delivery vehicle for locally delivering thecompound to dermal or mucosal cells of skin, scalp, mouth,nasoesophageal, gastrointestinal or urogenital system, wherein Formula Iis:

wherein: each Z is independently A or R¹; each A is independently:

J is a single bond or —CH(Y)—; X is D or —R²-D; Y is H, alkyl, or R³-D;D is —OH, —SH, —SR⁴, or —NR⁴R⁵; each R¹ is independently C₃₋₈ alkylene;each R², R³, R⁶, and R⁷ is independently C₁₋₆ alkylene; R⁴ is H or loweralkyl; R⁵ is H, lower alkyl, or —R⁶-D; Q is H, lower alkyl, or —R⁷—SR⁴;k is an integer from 2 to about 16; or a stereoisomer, prodrug,pharmaceutically acceptable salt, or mono or polyprotonated acid saltthereof.